IgG1 fusion protein activates CD8+ T cells in vivo

Clinical Immunology 116 (2005) 65 – 76 www.elsevier.com/locate/yclim

A soluble divalent class I MHC/IgG1 fusion protein activates $ CD8+ T cells in vivo Brenna Careya,b, Monica DeLaya, Jane E. Strasserc, Claudia Chalkc, Kristen Dudley-McClainc, Gregg N. Milligand, Hermine I. Brunnera, Sherry Thorntona, Raphael Hirsche,T a William S. Rowe Division of Rheumatology, Children’s Hospital Medical Center, Cincinnati, OH 45229, USA Graduate Program in Molecular and Developmental Biology, University of Cincinnati, Cincinnati, OH 45229, USA c Division of Infectious Diseases, Children’s Hospital Medical Center, Cincinnati, OH 45229, USA d Department of Pediatrics, University of Texas Medical Branch, Galveston, TX 77555, USA e Division of Rheumatology, Children’s Hospital of Pittsburgh, University of Pittsburgh, School of Medicine, 3705 Fifth Avenue, Pittsburgh, PA 15213, USA b

Received 17 July 2003; accepted with revision 22 February 2005 Available online 7 April 2005

Abstract CD8+ T lymphocytes recognize tumor and viral antigens bound to class I major histocompatibility complexes (MHC). Tumors and viruses may evade detection by preventing antigen presentation. The present study was designed to determine whether a soluble divalent fusion protein, containing the extracellular domains of a class I MHC molecule fused to h2-microglobulin and the constant domains of IgG1, could induce an immune response in vivo. Administration to mice of the fusion protein loaded with a tumor peptide induced peptide-specific T cell activation and retarded tumor growth. Administration of the fusion protein loaded with a glycoprotein B (gB) peptide derived from herpes simplex virus type 1 (HSV-1) induced gB-specific cytotoxic T lymphocytes and protected mice from a lethal HSV-1 challenge. These data suggest that antigen-loaded MHC/IgG fusion proteins may enhance T cell immunity in conditions where antigen presentation is altered. D 2005 Elsevier Inc. All rights reserved. Keywords: MHC; T lymphocytes; CTL; Tumor immunity; Viral immunity; Vaccination

Introduction CD4+ and CD8+ T cells play important roles in recognizing tumors and virally infected cells. CD4+ T cells recognize antigen in the context of class II major histocompatibility complexes (MHC) and secrete cytokines, such as IL-2 and IFN-g, that aid in the activation of CD8+ T cells. When CD8+ T cells recognize antigen in the context of class I MHC molecules in the presence of cytokines, they can become cytolytic T lymphocytes (CTL) and eliminate the

This work was supported, in part, by NIH grants AI34958 and AR47363. T Corresponding author. Division of Rheumatology, Children’s Hospital of Pittsburgh, University of Pittsburgh, School of Medicine, 3705 Fifth Avenue, Pittsburgh, PA 15213, USA. Fax: +1 412 692 5054. E-mail address: [email protected] (R. Hirsch). $

1521-6616/$ - see front matter D 2005 Elsevier Inc. All rights reserved. doi:10.1016/j.clim.2005.02.013

antigen presenting cell. Most tumors, especially solid tumors, are MHC class I+ and class II ; therefore, CD8+ CTLs are the major effector cells capable of directly lysing these tumor cells [1]. CD8+ CTLs also play an important role in recovery from primary viral infections, such as Herpes Simplex Virus (HSV), and in controlling the spread of HSV to the central nervous system [2]. Tumors have developed a variety of mechanisms to escape immune surveillance [3]. Some tumors lack expression of costimulatory molecules or proteins involved in antigen presentation to CD8+ T cells. T cell recognition of antigen in the absence of costimulatory signals results in the induction of T cell anergy, leading to anti-tumor T cells that are tolerant to the tumor antigen [3–5]. Tumors may lose expression of surface MHC molecules or immunogenic tumor antigens due to a decrease in transcription or translation of class I MHC molecules and h2 microglobulin

66

B. Carey et al. / Clinical Immunology 116 (2005) 65–76

(h2m) or due to other defects in antigen presentation [4,6– 16]. Because tumors can downregulate expression of MHC molecules or tumor antigens prior to the development of an effective anti-tumor immune response, investigators have attempted to use soluble MHC/peptide complexes to induce anti-tumor CD8+ T cell clones [17]. Activation of naRve HSV-specific CD8+ T cells may aid in controlling the initial viral infection of local epithelia, as well as decreasing the amount of virus that can traffic to the ganglia to establish a latent infection. HSV expresses several envelope glycoproteins, such as glycoprotein B (gB), that mediate the entry of the virus into host cells and trafficking of the virus from the site of infection to the central nervous system [18]. The immunodominant epitope recognized by HSV-specific CTLs in mice is gB498–505 (SSIEFARL) [2,19–22]. Adoptive transfer of HSV-specific CTL prior to an HSV-1 challenge decreases the amount of HSV detected in the footpad of infected mice [23]. Administration of vaccinia virus-expressing gB can induce the generation of anti-HSV CTL [24] and protect mice from lethal intraperitoneal [25,26] or corneal infection with HSV-1 [26,27]. Such studies suggest that the induction of an adequate primary response to HSV prior to infection may lead to the generation of a more effective secondary immune response capable of controlling a subsequent HSV infection. The use of soluble MHC/peptide complexes to activate CTL in vivo has obvious theoretical appeal. However, soluble MHC/peptide complexes have not been effective at inducing immunity in vivo, likely due to at least three reasons. First, multivalent T cell receptor (TCR) cross-linking appears to be necessary for effective T cell activation, as monovalent engagement of TCR, either with anti-TCR monoclonal antibodies [28] or with soluble class I MHC molecules [29–31], fails to induce signaling. Second, T cell activation requires a costimulatory signal, in addition to that mediated via the TCR [32–35]. This costimulatory signal is generally delivered by the antigen presenting cell; however, in the case of soluble class I MHC molecules, this second signal is absent. Third, soluble class I molecules are likely to have short half-lives in vivo. To address these limitations, we generated a divalent, soluble class I molecule by fusing the extracellular domains of murine H-2Kb to the constant domains of the IgG1 antibody. The molecule was designed to be divalent and to bind Fc receptor (FcR) bearing cells, allowing for multivalent TCR cross-linking and T cell activation. Incubation of this molecule with antigenic peptide activated antigenspecific T cells in vitro and induced the upregulation of CD69 expression on the surface of CD8+ T cells in vivo [36]. However, full T cell activation and induction of immunity was not achieved. In order to increase the stability of the fusion protein molecule, we have covalently attached h2-microglobulin (h2m) to the amino-terminus of the class I MHC region. The resulting molecule has superior biologic activity in vivo and

is able to fully-activate CD8+ T cells and induce anti-tumor and anti-viral immunity.

Materials and methods Mice C57BL/6 female mice were purchased from the Jackson Laboratory (Bar Harbor, ME). OT-I mice transgenic for an ovalbumin257–264 (OVA)-specific, H-2Kb-restricted TCR were obtained from F. Carbone and W. Heath. The OT-I mice were generated on a C57BL/6 background and carry the TCR Va2 and Vh5 transgenes [37]. Heterozygous OT-I male mice were mated to wild-type C57BL/6 females. OT-I heterozygotes were identified by staining peripheral blood lymphocytes with fluorescein isothiocyanate (FITC)-conjugated anti-Vh5 and phycoerythrin (PE)-conjugated anti-CD8 (BD PharMingen, La Jolla, CA) and analyzing the cells by flow cytometry using a FacsCalibur (Becton Dickinson Immunocytometry Systems, San Jose, CA). Heterozygous mice were readily identifiable, as virtually all transgenic T cells express the Vh5 TCR and are CD8+. Mice were cared for according to American Association for the Accreditation of Laboratory Animal Care guidelines. Cell culture and propagation of virus EG.7 is derived from the C57BL/6 thymoma cell line, EL.4, by transfection with chicken ovalbumin cDNA [38] and expresses OVA on its cell surface. EG.7 and EL.4 cells were maintained in complete RPMI (Gibco Invitrogen Corporation, Grand Island, NY) with 10% Fetal Calf Serum (FCS) in a humidified 5% CO2 chamber. Virus stocks of HSV-1 McKrae were animal passed and then cultured on Vero cell monolayers and prepared by two cycles of freeze/thaw and removal of cellular debris by centrifugation. Viral stocks were stored at 808C and samples were titrated on Vero cell monolayers. Peptides OVA (SIINFEKL) and gB (SSIEFARL) peptides were purchased from the University of North Carolina Program in Molecular Biology and Biotechnology Micro Protein Chemistry Facility (Chapel Hill, NC). SIINFEKL and SSIEFARL are the immunodominant epitopes derived from either chicken ovalbumin residues 257–264 or Herpes simplex virus glycoprotein B residues 498–505, respectively. Both peptides can be presented to TCR by murine H2Kb [2,39]. Divalent MHC/IgG1 fusion protein H-2Kb-IgG1 was constructed by fusing the extracellular domains of H-2Kb to the hinge, CH2 and CH3 domains of

B. Carey et al. / Clinical Immunology 116 (2005) 65–76

mouse IgG1, as previously described [36]. The DNA sequence encoding h2m was ligated to the amino-terminus of H-2Kb-IgG1 with a polyglycine tether, (GGGGS)4, to generate H-2Kb-h2m-IgG1. The product was gel purified, ligated into the expression vector, pCMV4, and transiently transfected into COS-7 cells. Cells were pulsed with 35Smethionine and supernatants were immunoprecipitated with sepharose 4B conjugated to the conformationallydependent anti-H-2Kb monoclonal antibody, Y3. Electrophoresis on 4–20% gradient gels showed a novel 180kilodalton (kDa) protein in supernatants from transfected cells, which reduced to a 90-kDa protein, demonstrating that the fusion protein is a homodimer composed of two 90-kDa polypeptides, approximating the predicted size of the fusion protein. The fusion protein sequence was expressed in the baculovirus pFASTBAC expression system (Invitrogen, Carlsbad, CA). Sf9 insect cells were infected with baculovirus. Five days following infection, media containing fusion protein was harvested and stored at 48C.

67

cells were added to each well. After 24 h, the splenocytes were pulsed with [3H] TdR, 1 ACi/well. Eighteen hours later, the cells were harvested and [3H] TdR incorporation was measured on a TopCount-NXT microplate scintillation counter (Packard, Meriden, CT). All samples were assayed in triplicate. Flow cytometry OT-I mice were injected intraperitoneally (i.p.) with 1 ml of culture supernatant containing peptide-loaded fusion protein or peptide alone (5 Ag/ml). One or 7 days later, spleens were harvested and single cell suspensions were generated. Cells were stained with PE-conjugated anti-CD8 and either FITC-conjugated anti-CD25 or anti-CD44 (BD Pharmingen, La Jolla, CA). Multi-color flow cytometry was performed using a FacsCalibur (Becton Dickinson Immunocytometry Systems, San Jose, CA), gating on CD8+ T cells. In vivo treatment and proliferation assay

ELISA for the detection of peptide-loaded fusion protein Ninety-six-well, flat-bottom enzyme-linked immunosorbent assay (ELISA) plates (Becton Dickinson Labware, Franklin Lakes, NJ) were coated with anti-IgG1 (Caltag Laboratories, Inc., San Francisco, CA) at a concentration of 5 Ag/ml overnight at 48C. The fusion protein was loaded with peptide overnight at 48C. The plates were washed with phosphate buffered saline (PBS)/Tween (1% Tween-20), blocked with 1% bovine serum albumin in PBS, washed again, and incubated in duplicate with peptide-loaded fusion protein. Unbound fusion protein was washed from the plate and biotinylated Y3 antibody (5 Ag/ml), recognizing peptide-loaded H-2Kb [40–43], was added. Plates were washed, followed by the addition of streptavidin-horseradish peroxidase (HRP) (Zymed Laboratories, Inc., San Francisco, CA). Plates were washed, developed with ABTS microwell peroxidase substrate system (Kirkegaard and Perry Laboratories, Gaithersburg, MD), and read at 405 nm with a Kinetic Microplate Reader (Molecular Devices, Sunnyvale, CA). In vitro OT-I spleen cell assay Ninety-six-well, flat-bottom plates were coated with antiIgG1 (Caltag Laboratories, Inc., San Francisco, CA) at a concentration of 10 Ag/ml overnight at 48C. At the same time, OVA or gB peptide (10 ng/ml) was incubated with culture supernatant containing fusion protein overnight at 48C. The plates were then washed with PBS, blocked with 1% bovine serum albumin in PBS for 1 h, washed again, and incubated for 4 h at 48C with 100 Al of culture supernatant containing peptide-loaded fusion protein or peptide alone. Plates were washed with PBS to remove unbound fusion protein and 2  105 OT-I mouse spleen

C57BL/6 mice were injected i.p. with 1 ml of 10 culture supernatant containing peptide-loaded fusion protein (5 Ag/ml of peptide), peptide alone, or media. Mice were simultaneously treated with 50 Ag LPS (Sigma, St. Louis, MO) subcutaneously (s.c.) to induce expression of cytokines and upregulation of costimulatory molecules [44]. For assessment of a proliferative response, splenocytes were harvested 7 days later and incubated (4  105 cells) with OVA or gB peptide (0.1 Ag/ml). Proliferation was assessed at 72 h, as described above. Fold stimulation was determined by dividing the average counts of [3H] TdR incorporated by splenocytes stimulated with peptide by the average counts of [3H] TdR incorporated by splenocytes incubated in media alone. All samples were assayed in triplicate. Cytotoxic T lymphocyte assay Cytotoxicity assays were performed as previously described [45]. Briefly, C57BL/6 mice were immunized with peptide-loaded fusion protein, peptide alone, or PBS. Seven days later, 4  106 spleen cells from treated mice were stimulated in 24-well plates with 4  106 irradiated (2500 rads) syngeneic spleen cells pulsed with gB peptide (10 Ag/ml), in the presence of 10 units/ml of recombinant IL-2. After 5 days incubation at 378C, effector cells were harvested and examined in a 4-h 51Cr release assay against OVA- or gB-pulsed EL.4 target cells (a C57BL/6-derived thymoma cell line). Percent lysis of OVA-pulsed target cells was subtracted from percent lysis of gB-pulsed targets to determine specific lysis. Each assay was performed in triplicate and the results were averaged. Percent specific lysis = (experimental release spontaneous release)/ (maximum release spontaneous release)  100.

68

B. Carey et al. / Clinical Immunology 116 (2005) 65–76

Tumor challenge

Results

For tumor studies, 7 days after injection of LPS and fusion protein or peptide, as previously described above, mice were anesthetized, shaved with clippers, and injected subcutaneously with 2  106 EG.7 tumor cells. Tumor volumes were measured every other day using vernier calipers beginning on day 7 following tumor challenge.

MHC/IgG1 fusion protein can be loaded with exogenous peptide

Tumor antigen assay OT-I effector spleen cells (4  106) were induced by stimulating OT-I spleen cells for 5 days with 4  106 irradiated (2500 rads) syngeneic C57BL/6 spleen cells pulsed with OVA peptide (10 Ag/ml), in the presence of 10 units/ml of recombinant IL-2. After 5 days, single cell suspensions (1  106) of EG.7 tumors that had been excised from C57BL/6 mice were labeled with 300 ACi Na51CrO4 for 1 h at 378C. Following labeling, half of the tumor cells were pulsed with OVA peptide (10 Ag/ml) for 1 h. OT-I effector cells were examined in a 4-h 51Cr release assay against unpulsed and OVA-pulsed tumor cells. Each assay was performed in triplicate and the results were averaged. Percent specific lysis = (experimental release spontaneous release)/(maximum release spontaneous release)  100. Maximum release and spontaneous release were determined by adding 0.1 N HCL or media, respectively, to target tumor cells. Herpes infection Mice were immunized i.p. with peptide-loaded fusion protein, peptide alone, or PBS. Mice were simultaneously treated with 50 Ag LPS subcutaneously. Fourteen days later, mice were challenged i.p with 5  107 pfu/ml (1LD50) HSV-1 McKrae. Survival of mice was monitored for 21 days following viral challenge.

To determine whether the fusion protein could bind peptide, culture supernatants containing the fusion protein were incubated with varying concentrations of peptide. Addition of peptide led to a dose-dependent increase in binding of the conformationally-dependent anti-H-2Kb monoclonal antibody, Y3 [40–43] (Fig. 1), demonstrating that the fusion protein was conformationally intact. For reasons that are unclear, we were unable to purify the fusion protein without losing conformation, despite exhaustive efforts. Therefore, all further studies were done with supernatants, using appropriate controls to ensure specificity. Divalent MHC/IgG1 fusion protein induces antigen-specific T cell activation in vitro We first sought to determine whether the fusion protein could activate CD8+ T cells in vitro. For this purpose, we utilized the OT-I mouse in which all T cells are CD8+ and recognize the OVA peptide in the context of H-2Kb. OT-I spleen cells were incubated with OVA-loaded fusion which had been immobilized on plastic wells by cross-linking with anti-IgG1. A strong proliferative response was observed in response to OVA-loaded fusion protein, compared to fusion protein loaded with the control gB peptide, unloaded fusion protein, or OVA peptide alone (Fig. 2). Thus, the fusion protein induced in vitro proliferation of T cells in an antigenspecific manner.

Statistical analysis The duration of tumor-free survival of mice immunized with OVA-loaded fusion protein was compared to that of the controls (gB-loaded fusion protein, OVA peptide, and gB peptide) via life table analysis and COX proportional hazard modeling. The size of the tumors between days 7 through 21 post inoculation with EG.7 tumor cells was compared for important differences using both parametric analysis of variance (ANOVA) and non-parametric ANOVA (Kruskal–Wallis test). Post hoc testing was done using Tukey’s method (parametric ANOVA) and Median test (non-parametric ANOVA). The advantage of parametric testing is that the exact size of the tumors is used, while non-parametric analysis is based on ranking the size of the tumors. However, since our tumor volumes were highly skewed, parametric analysis was not suitable for this data.

Fig. 1. MHC/IgG1 fusion protein can be loaded with exogenous peptide. gB peptide was incubated at 48C overnight with fusion protein. gBloaded fusion protein was then immobilized on 96-well plates with antiIgG1 and was detected with the conformationally-dependent anti-H-2Kb monoclonal antibody, Y3. Each point represents the mean of two individual values.

B. Carey et al. / Clinical Immunology 116 (2005) 65–76

69

Fig. 2. OVA-loaded fusion protein induces antigen-specific T cell proliferation of OT-I splenocytes. Fusion protein was loaded with OVA or gB peptide (10 ng/ ml) and immobilized onto anti-IgG1-coated 96-well plates. Unbound fusion protein and peptide were removed and OT-I spleen cells were added. Proliferation of OT-I splenocytes is depicted as counts and is depicted as the mean and SEM of triplicate measures. Graph is representative of three individual experiments.

Divalent MHC/IgG1 fusion protein activates OT-I T cells in vivo Having determined that the fusion protein could activate T cells in vitro in an antigen-specific manner, we next asked whether the same might be true under in vivo conditions. OT-I mice were injected i.p. with OVA-loaded fusion protein, gB-loaded fusion protein, or OVA peptide alone. Spleens were harvested after 1 and 7 days and the expression of T cell activation markers on CD8+ T cells was analyzed via flow cytometry. Upregulation of CD25 (IL-2 receptor) was observed 24 h following administration of OVA-loaded fusion protein (Fig. 3A). Upregulation of the memory marker, CD44, was observed 7 days after immunization with the OVA-loaded fusion protein (Fig. 3B). OVA peptide alone or fusion protein loaded with the control gB peptide failed to induce T cell activation, demonstrating the specificity of the response. Divalent MHC/IgG1 fusion protein primes T cells in vivo Having demonstrated in vivo T cell activation, we next sought to determine whether the fusion protein

could fully prime T cells in vivo in an antigen-specific manner in wild-type mice. C57BL/6 mice were injected with OVA-loaded fusion protein, gB-loaded fusion protein, OVA peptide alone, or gB peptide alone. Since the fusion protein is composed of a class I molecule, its specificity is limited to CD8+ T cells. Therefore, to ensure sufficient T cell help, mice also received a subcutaneous injection of 50 Ag of LPS to induce secretion of helper cytokines [44]. After 7 days, spleen cells from treated mice were analyzed for the ability to proliferate in response to in vitro restimulation with OVA or gB. Spleen cells from mice injected with the OVA-loaded fusion protein proliferated in response to OVA, but not gB (Fig. 4). Conversely, spleen cells from mice injected with the gB-loaded fusion protein proliferated in response to gB, but not OVA. The stimulation index of approximately 1.5 and 2 for OVA-loaded and gB-loaded fusion protein, while low, was a consistent finding in multiple repetitions of this experiment. In each case, gB-loaded fusion protein induced a stronger proliferative response than OVA-loaded fusion protein. Immunization with OVA peptide alone or gB peptide alone did not prime T cells in vivo.

Fig. 3. OVA-loaded fusion protein induces upregulation of T cell activation markers in vivo. OT-1 mice were administered peptide-loaded fusion protein (5 Ag/ ml peptide) or peptide alone. Splenocytes were removed 1 day (A) or 7 days (B) later and analyzed by flow cytometry for expression of CD25 and CD44. Histograms represent expression following gating on CD8+ spleen cells.

70

B. Carey et al. / Clinical Immunology 116 (2005) 65–76

Fig. 4. Peptide-loaded fusion protein primes T cells in vivo. C57BL/6 mice were administered peptide-loaded fusion protein (5 Ag/ml peptide) or peptide alone. Splenocytes were removed after 7 days and stimulated in vitro with OVA or gB (0.1 Ag/ml). Stimulation index represents the proliferative response to peptide divided by the background response to media alone and is depicted as the mean of triplicate measures. Each sample was assayed in triplicate. *P b 0.05 by Student’s t test.

MHC/IgG1 fusion protein induces the generation of antigen-specific CTL To determine whether in vivo administration of the gBloaded fusion protein could induce gB-specific CTL, C57BL/6 mice were immunized with peptide-loaded fusion protein, gB peptide alone, or PBS, plus 50 Ag of LPS. After 7 days, spleen cells from treated mice were analyzed for the presence of gB-specific CTL. Spleen cells from mice immunized with gB-loaded fusion protein were able to specifically lyse gB-pulsed targets (Fig. 5). Mice immunized with OVA-loaded fusion protein, gB alone, or PBS were unable to generate gB-specific CTL. Therefore, the fusion protein was able to induce the generation of gB-specific CTL in vivo. We were consistently able to induce CTL against gB using gB-loaded fusion protein, but we were not able to induce CTL against OVA following administration

of OVA-loaded fusion protein. This did not rule out the possibility that anti-OVA CTLs were present in vivo at a precursor frequency too low to detect. Divalent MHC/IgG1 fusion protein delays the growth of a CD8-dependent tumor To determine whether the fusion protein could be used as a vaccine to immunize mice against tumors, C57BL/6 mice (10/group) were immunized with OVA-loaded fusion protein. Control mice received gB-loaded fusion protein, OVA peptide, or gB peptide. All mice also received a subcutaneous injection of 50 Ag of LPS. Seven days later, mice were challenged with the EG.7 tumor cell line by injection of 2  106 cells s.c. in the flank. EG.7 cells grow in C57BL/6 mice unless the mice have functional anti-OVA CD8+ CTL [38]. Mice immunized with the OVA-loaded

Fig. 5. MHC/IgG1 fusion protein induces antigen-specific CTL. Mice were immunized with peptide-loaded fusion protein, gB alone, or PBS. Seven days later, mice were sacrificed and spleen cells were stimulated for 5 days with gB-pulsed syngeneic spleen cells. Effector cells were harvested and examined in a 4-h 51 Cr release assay against gB-pulsed EL.4 target cells. Percent lysis of OVA-pulsed target cells was subtracted from lysis of gB-pulsed targets to determine specific lysis. Results are representative of two individual experiments.

B. Carey et al. / Clinical Immunology 116 (2005) 65–76

fusion protein demonstrated a significant delay ( P b 0.05 by life table analysis and COX proportional hazard modeling) in the onset of detectable tumors (Fig. 6A). In addition, a significant decrease in tumor size ( P b 0.05 on days 13, 15, 17, and 19 by Kruskal–Wallis test and Median test) was observed, compared to controls (Fig. 6B). This protective effect was reproducible in 2 additional repetitions of this experiment (data not shown). Mice immunized with OVA-loaded fusion protein eventually developed tumors. To test the possibility that this loss of protection might be due to outgrowth of antigenloss tumor cell variants [46–48], mice were sacrificed 17 days following tumor injection and tumors developing in the mice immunized with OVA-loaded or the control gB-loaded fusion protein were isolated. EG.7 cells derived from these tumors were labeled with 51Cr and used as target cells in a 4-h CTL assay using OVA-specific CTL effectors generated form OT-I spleens. EG.7 cells derived from the tumors had lost the ability to be lysed by OVA-specific CTL (Fig. 7A). Lysis was restored by pulsing the targets with OVA peptide (Fig. 7B). These studies suggest that the eventual growth of tumors from mice immunized with OVA-loaded fusion

71

protein may have been due to outgrowth of OVA-negative tumor variants. Divalent MHC/IgG1 fusion protein protects against lethal HSV-1 challenge To determine whether in vivo administration of the gBloaded fusion protein could protect mice from HSV-1 infection, C57BL/6 mice were immunized with peptideloaded fusion protein, gB peptide alone, or PBS, plus 50 Ag of LPS. Fourteen days later, mice were challenged with a lethal dose of HSV-1 McKrae. Mice immunized with gBloaded fusion protein were protected from HSV-1, whereas mice immunized with OVA-loaded fusion protein or gB peptide alone were not (Fig. 8).

Discussion Induction of T cell immunity using soluble MHC molecules has been a theoretically appealing approach that has been difficult to achieve experimentally. In the present

Fig. 6. OVA-loaded fusion protein delays the onset (A) and growth (B) of EG.7 tumors. Mice were immunized as indicated (10 mice/group) and challenged 7 days later with 2  106 EG.7 tumor cells. Tumor onset was significantly delayed in mice immunized with the OVA-loaded fusion protein compared to control mice (P b 0.05 by life table analysis). Tumor volumes are represented as the mean and SEM. *P b 0.05 by Kruskal–Wallis ANOVA.

72

B. Carey et al. / Clinical Immunology 116 (2005) 65–76

Fig. 7. Outgrowth of OVA-negative tumor variants following in vivo growth of EG.7 tumor cells. Effector CTL were generated from OT-I transgenic spleen cells, as described in Materials and methods. EG.7 tumors were isolated from mice immunized with OVA-loaded fusion protein (E) or HSV-loaded fusion protein (n) 17 days following tumor challenge. Tumor cells were prepared as single cell suspensions, labeled with 51Cr and used as targets in a 4-h 51Cr release assay (A). Some EG.7 targets were pulsed with OVA prior to the assay (B). Percent lysis was determined at the effector:target ratios indicated. EG.7 tumor cells grown in culture (.) served as a positive control target.

study, we utilized a soluble MHC molecule designed to multivalently cross-link TCR and to bind FcR-bearing accessory cells to induce T cell activation and tumor immunity. To our knowledge, this is the first study to achieve full in vivo activation of T cells using a soluble MHC molecule. The demonstration that T cells of OT-I mice and C57BL/ 6 mice could be activated in an antigen-specific manner by in vivo administration of OVA- or gB-loaded fusion protein suggests the likelihood that the fusion protein inhibited tumor growth and controlled viral growth via induction of CTL. We were unable to detect the presence of OVA-

specific CTL in the spleens of C57BL/6 mice administered OVA-loaded fusion protein, using the standard 51Cr release assay. We were able to induce gB-specific CTL in spleens of C57BL/6 mice immunized with gB-loaded fusion protein. It is likely that OVA-specific CD8+ T cells were present at undetectable levels in the tumors in sufficient numbers to inhibit initial tumor growth until the outgrowth of antigenloss variants. A recent study by Goldberg et al. supports this possibility [49]. The authors demonstrated that OT-I T cells, adoptively transferred into C57BL/6 mice, could be activated to express CD44 by OVA-loaded H-2Kb that had been immobilized on microspheres. When administered

Fig. 8. MHC/IgG1 fusion protein protects against lethal HSV-1 challenge. Wild-type mice were immunized with peptide-loaded fusion protein, gB alone, or PBS (12 mice/group). Fourteen days later, mice were challenged with 5  107 pfu/ml HSV-1 McKrae. The survival of the mice was monitored for 21 days following HSV-1 challenge. Mice immunized with gB-loaded fusion protein demonstrated significant protection ( P b 0.05 by log rank test). Results are representative of three individual experiments.

B. Carey et al. / Clinical Immunology 116 (2005) 65–76

alone, the microspheres caused only weak clonal expansion of OT-I T cells in the spleen. However, when administered with EG.7 cells, the microspheres caused a rapid increase in the number of OT-I T cells at the site of the growing tumor. The OVA-loaded fusion protein was able to protect mice against tumor growth until the tumor no longer presented the tumor antigen, OVA, on its surface. Tumor cells isolated from wild-type mice could be pulsed with OVA peptide and lysed by in vitro stimulated OT-I CTLs (Fig. 7A), indicating that the EG.7 tumor cells isolated from these mice expressed sufficient MHC molecules on their surface, but over time lost presentation of the OVA tumor antigen. It is unlikely that the loss of OVA presentation was due to selective pressure by the immune system because tumor cells isolated from mice immunized with the gB-loaded fusion protein also lost OVA presentation. The melanoma cell line, MU, has been shown to downregulate expression of the melanoma antigen, Melan-A/MART-1, by expressing the cytokine oncostatin M, which signals via the gp130 cell surface receptor and suppresses the promoter activity of the antigen when the cells are grown at high density [15]. The EG.7 tumor cell line may express a similar factor that is capable of downregulating OVA presentation or a novel mechanism of antigen downregulation may be employed by these tumor cells. The mechanism(s) the EG.7 tumor cells utilize to escape detection by the immune system needs to be further examined. In 2001, O’Herrin et al. demonstrated that a dimeric fusion protein similar to ours was able to induce early activation of T cells in vitro, but in vivo administration of this dimer suppressed CD8+ T cell effector function [50]. In vitro stimulation of 2C transgenic T cells, which express a QL9-specific, H-2Ld-restricted TCR, with this dimer resulted in modulation of the TCR, upregulation of CD69 expression, and phosphorylation of TCR ~-chain and ZAP70. 2C transgenic T cells activated in vitro by the peptide/ MHC dimer were unable to lyse peptide-pulsed targets in an in vitro 51Cr release assay and were unable to protect against tumor challenge when adoptively transferred into RAG2deficient mice. The discrepancies in the conclusions from our study and that of O’Herrin et al. may be due to a number of factors. The overall structures of the fusion proteins differ. Their MHC/Ig dimer was composed of the extracellular region of a class I MHC molecule linked to a scaffold of an entire immunoglobulin molecule, as opposed to the hinge and constant domains present in our molecule. In addition, the class I MHC alleles and the immunogenic peptides used were different. The methods of priming the T cells (in vivo versus in vitro), the type of T cells activated (wild-type versus transgenic), and the mice used for the tumor experiments (wild-type versus RAG2 deficient) were also different between the two studies. Perhaps most critical, we were only able to achieve an effective immune response by coadministering LPS at the time of fusion protein administration to induce the

73

expression of cytokines and upregulation of costimulatory molecules. An elegant in vivo model system recently developed by Jenkins and colleagues has helped to elucidate some of the parameters determining whether an antigenic challenge results in T cell priming or in T cell tolerance [44,51,52]. These studies demonstrate that, in naive T cells in vivo, signal transduction mediated by TCR and CD28 engagement may not be sufficient for the generation of immunologic memory. Administration of antigenic peptide alone induces transient B7-dependent T cell activation in lymph nodes and spleen, followed by the disappearance of most T cells. Remaining cells are hyporesponsive to restimulation and immunologic memory is not produced [51]. However, if antigen is presented in the context of an inflammatory process, enhanced numbers of T cells accumulate, migrate into B cell-rich follicles, acquire the capacity to produce IFN-g, and become primed memory cells. This inflammation can be mimicked by LPS or cytokines such as TNF-a and IL-1. Numerous other anti-tumor vaccine strategies are being explored, including loading dendritic cells with tumor antigens [53–57], vaccination with plasmid DNA encoding tumor antigens [58–61], and the adoptive transfer of transgenic CD8+ T cells specific for the immunodominant epitope of the tumor cell [53,62–64]. In some cases, these vaccinations induced autoimmune reactions [6,65], they are labor intensive, and costly [66]. Furthermore, vaccination with DNA vectors may result in the transfection of somatic cells that lack expression of costimulatory molecules necessary to activate T cells, resulting in anergized antitumor T cells. In humans, adoptive cellular immunotherapy involving the reinfusion of patient-derived effector cells has been less effective due to suboptimal response rates, limited response durations, and extreme toxicity associated with high doses of IL-2, necessary for the survival of transferred T cells [62,67–69]. The development of an HSV vaccine is of high priority, given the increasing prevalence of individuals infected with HSV, the severity of the disease in neonates and immunocompromised individuals, and the lack of an effective treatment. Initial vaccine strategies focused on immunization with attenuated HSV. However, the development of molecular biology techniques has allowed the development of many experimental vaccine strategies. These include immunization with purified protein [21,70,71] as well as the delivery of HSV antigens by adenovirus [72], vaccinia virus [24–27,73–75], and plasmid DNA [71,76]. In many cases, these vaccination strategies utilize the same antigen presentation pathway the virus has evolved to evade. HSV has evolved a variety of strategies to subvert the immune system and establish a long-lasting infection. One such strategy is the expression of the viral protein, ICP-47, which prevents the presentation of viral antigen to CD8+ T lymphocytes by inhibiting the transport of peptides into the endoplasmic reticulum, thus preventing peptide loading into class I MHC molecules [77]. In

74

B. Carey et al. / Clinical Immunology 116 (2005) 65–76

addition, virus-based vaccines may induce an inflammatory response which may damage host tissue [72,73,78,79]. Vaccination with DNA vectors may result in the transfection of somatic cells that lack costimulatory molecules, resulting in the induction of T cell anergy, as opposed to T cell activation [80]. Soluble MHC/IgG fusion proteins like the one described in the present study can be constructed to be specific for the host, thereby limiting potential immune reactions to the immunogen. While it is not known whether such fusion proteins could be used in a therapeutic setting to treat established cancers or vaccinate against viral infections, they might be useful as a tool to study the potential of specific peptides and altered peptides to induce T cell immunity.

Acknowledgments

[11]

[12]

[13]

[14]

[15]

We thank Kristi Jennings for technical support and Drs. E. Choi, R. Colbert, J. Katz, and D. Wiginton for helpful discussion and critical review of the manuscript.

References [1] A.L. Marzo, B.F. Kinnear, R.A. Lake, J.J. Frelinger, E.J. Collins, B.W. Robinson, B. Scott, Tumor-specific CD4+ T cells have a major bpostlicensingQ role in CTL mediated anti-tumor immunity, J. Immunol. 165 (2000) 6047 – 6055. [2] T. Hanke, F.L. Graham, K.L. Rosenthal, D.C. Johnson, Identification of an immunodominant cytotoxic T-lymphocyte recognition site in glycoprotein B of herpes simplex virus by using recombinant adenovirus vectors and synthetic peptides, J. Virol. 65 (1991) 1177 – 1186. [3] T.A. Cordaro, K.E. de Visser, F.H. Tirion, Y.M. Graus, J.B. Haanen, D. Kioussis, A.M. Kruisbeek, Tumor size at the time of adoptive transfer determines whether tumor rejection occurs, Eur. J. Immunol. 30 (2000) 1297 – 1307. [4] N. Dalyot-Herman, O.F. Bathe, T.R. Malek, Reversal of CD8+ T cell ignorance and induction of anti-tumor immunity by peptide-pulsed APC, J. Immunol. 165 (2000) 6731 – 6737. [5] A.F. Ochsenbein, S. Sierro, B. Odermatt, M. Pericin, U. Karrer, J. Hermans, S. Hemmi, H. Hengartner, R.M. Zinkernagel, Roles of tumour localization, second signals and cross priming in cytotoxic Tcell induction, Nature 411 (2001) 1058 – 1064. [6] D. Jager, E. Jager, A. Knuth, Immune responses to tumour antigens: implications for antigen specific immunotherapy of cancer, J. Clin. Pathol. 54 (2001) 669 – 674. [7] C.M. D’Urso, Z.G. Wang, Y. Cao, R. Tatake, R.A. Zeff, S. Ferrone, Lack of HLA class I antigen expression by cultured melanoma cells FO-1 due to a defect in B2m gene expression, J. Clin. Invest. 87 (1991) 284 – 292. [8] D.C. Bicknell, A. Rowan, W.F. Bodmer, Beta 2-microglobulin gene mutations: a study of established colorectal cell lines and fresh tumors, Proc. Natl. Acad. Sci. U. S. A. 91 (1994) 4751 – 4756. [9] D.J. Hicklin, D.V. Dellaratta, R. Kishore, B. Liang, T. Kageshita, S. Ferrone, Beta2-microglobulin gene mutations in human melanoma cells: molecular characterization and implications for immune surveillance, Melanoma Res. 7 (Suppl. 2) (1997) S67 – S74. [10] D.J. Hicklin, Z. Wang, F. Arienti, L. Rivoltini, G. Parmiani, S. Ferrone, beta2-Microglobulin mutations, HLA class I antigen loss,

[16]

[17]

[18]

[19]

[20]

[21]

[22]

[23]

[24]

[25]

and tumor progression in melanoma, J. Clin. Invest. 101 (1998) 2720 – 2729. R. Benitez, D. Godelaine, M.A. Lopez-Nevot, F. Brasseur, P. Jimenez, M. Marchand, M.R. Oliva, N. van Baren, T. Cabrera, G. Andry, C. Landry, F. Ruiz-Cabello, T. Boon, F. Garrido, Mutations of the beta2microglobulin gene result in a lack of HLA class I molecules on melanoma cells of two patients immunized with MAGE peptides, Tissue Antigens 52 (1998) 520 – 529. F.M. Marincola, E.M. Jaffee, D.J. Hicklin, S. Ferrone, Escape of human solid tumors from T-cell recognition: molecular mechanisms and functional significance, Adv. Immunol. 74 (2000) 181 – 273. M.J. Maeurer, S.M. Gollin, D. Martin, W. Swaney, J. Bryant, C. Castelli, P. Robbins, G. Parmiani, W.J. Storkus, M.T. Lotze, Tumor escape from immune recognition: lethal recurrent melanoma in a patient associated with downregulation of the peptide transporter protein TAP-1 and loss of expression of the immunodominant MART1/Melan-A antigen, J. Clin. Invest. 98 (1996) 1633 – 1641. N. Gervois, Y. Guilloux, E. Diez, F. Jotereau, Suboptimal activation of melanoma infiltrating lymphocytes (TIL) due to low avidity of TCR/MHC–tumor peptide interactions, J. Exp. Med. 183 (1996) 2403 – 2407. J.T. Kurnick, T. Ramirez-Montagut, L.A. Boyle, D.M. Andrews, F. Pandolfi, P.J. Durda, D. Butera, I.S. Dunn, E.M. Benson, S.J. Gobin, P.J. van den Elsen, A novel autocrine pathway of tumor escape from immune recognition: melanoma cell lines produce a soluble protein that diminishes expression of the gene encoding the melanocyte lineage melan-A/MART-1 antigen through down-modulation of its promoter, J. Immunol. 167 (2001) 1204 – 1211. E. Mottez, P. Langlade-Demoyen, H. Gournier, F. Martinon, J. Maryanski, P. Kourilsky, J.P. Abastado, Cells expressing a major histocompatibility complex class I molecule with a single covalently bound peptide are highly immunogenic, J. Exp. Med. 181 (1995) 493 – 502. J. Goldstein, H. Mostowsky, J. Tung, H. Hon, M. Brunswick, S. Kozlowski, Naive alloreactive CD8 T cells are activated by purified major histocompatibility complex class I and antigenic peptide, Eur. J. Immunol. 27 (1997) 871 – 878. W.H. Cai, B. Gu, S. Person, Role of glycoprotein B of herpes simplex virus type 1 in viral entry and cell fusion, J. Virol. 62 (1988) 2596 – 2604. L.A. Witmer, K.L. Rosenthal, F.L. Graham, H.M. Friedman, A. Yee, D.C. Johnson, Cytotoxic T lymphocytes specific for herpes simplex virus (HSV) studied using adenovirus vectors expressing HSV glycoproteins, J. Gen. Virol. 71 (1990) 387 – 396. R.H. Bonneau, S.R. Jennings, Herpes simplex virus-specific cytolytic T lymphocytes restricted to a normally low responder H-2 allele are protective in vivo, Virology 174 (1990) 599 – 604. R.H. Bonneau, L.A. Salvucci, D.C. Johnson, S.S. Tevethia, Epitope specificity of H-2Kb-restricted, HSV-1-, and HSV-2-cross-reactive cytotoxic T lymphocyte clones, Virology 195 (1993) 62 – 70. M.E. Wallace, R. Keating, W.R. Heath, F.R. Carbone, The cytotoxic T-cell response to herpes simplex virus type 1 infection of C57BL/6 mice is almost entirely directed against a single immunodominant determinant, J. Virol. 73 (1999) 7619 – 7626. R.H. Bonneau, S.R. Jennings, Modulation of acute and latent herpes simplex virus infection in C57BL/6 mice by adoptive transfer of immune lymphocytes with cytolytic activity, J. Virol. 63 (1989) 1480 – 1484. E. McLaughlin-Taylor, D.E. Willey, E.M. Cantin, R. Eberle, B. Moss, H. Openshaw, A recombinant vaccinia virus expressing herpes simplex virus type 1 glycoprotein B induces cytotoxic T lymphocytes in mice, J. Gen. Virol. 69 (1988) 1731 – 1734. E.M. Cantin, R. Eberle, J.L. Baldick, B. Moss, D.E. Willey, A.L. Notkins, H. Openshaw, Expression of herpes simplex virus 1 glycoprotein B by a recombinant vaccinia virus and protection of mice against lethal herpes simplex virus 1 infection, Proc. Natl. Acad. Sci. U. S. A. 84 (1987) 5908 – 5912.

B. Carey et al. / Clinical Immunology 116 (2005) 65–76 [26] H. Ghiasi, R. Kaiwar, A.B. Nesburn, S. Slanina, S.L. Wechsler, Expression of seven herpes simplex virus type 1 glycoproteins (gB, gC, gD, gE, gG, gH, and gI): comparative protection against lethal challenge in mice, J. Virol. 68 (1994) 2118 – 2126. [27] D.E. Willey, E.M. Cantin, L.R. Hill, B. Moss, A.L. Notkins, H. Openshaw, Herpes simplex virus type 1-vaccinia virus recombinant expressing glycoprotein B: protection from acute and latent infection, J. Infect. Dis. 158 (1988) 1382 – 1386. [28] R. Hirsch, J.A. Bluestone, L. DeNenno, R.E. Gress, Anti-CD3 F(abV)2 fragments are immunosuppressive in vivo without evoking either the strong humoral response or morbidity associated with whole mAb, Transplantation 49 (1990) 1117 – 1123. [29] J.P. Abastado, Y.C. Lone, A. Casrouge, G. Boulot, P. Kourilsky, Dimerization of soluble major histocompatibility complex–peptide complexes is sufficient for activation of T cell hybridoma and induction of unresponsiveness, J. Exp. Med. 182 (1995) 439 – 447. [30] J. McCluskey, L.F. Boyd, P.F. Highet, J. Inman, D.H. Margulies, T cell activation by purified, soluble, class I MHC molecules. Requirement for polyvalency, J. Immunol. 141 (1988) 1451 – 1455. [31] S.H. Herrmann, M.F. Mescher, The requirements for antigen multivalency in class I antigen recognition and triggering of primed precursor cytolytic T lymphocytes, J. Immunol. 136 (1986) 2816 – 2825. [32] M.K. Jenkins, C.A. Chen, G. Jung, D.L. Mueller, R.H. Schwartz, Inhibition of antigen-specific proliferation of type 1 murine T cell clones after stimulation with immobilized anti-CD3 monoclonal antibody, J. Immunol. 144 (1990) 16 – 22. [33] D.R. DeSilva, K.B. Urdahl, M.K. Jenkins, Clonal anergy is induced in vitro by T cell receptor occupancy in the absence of proliferation, J. Immunol. 147 (1991) 3261 – 3267. [34] M.E. Williams, C.M. Shea, A.H. Lichtman, A.K. Abbas, Antigen receptor-mediated anergy in resting T lymphocytes and T cell clones. Correlation with lymphokine secretion patterns, J. Immunol. 149 (1992) 1921 – 1926. [35] C.D. Gimmi, G.J. Freeman, J.G. Gribben, G. Gray, L.M. Nadler, Human T-cell clonal anergy is induced by antigen presentation in the absence of B7 costimulation, Proc. Natl. Acad. Sci. U. S. A. 90 (1993) 6586 – 6590. [36] C.M. Cullen, S.C. Jameson, M. DeLay, C. Cottrell, E.T. Becken, E. Choi, R. Hirsch, A divalent major histocompatibility complex/IgG1 fusion protein induces antigen-specific T cell activation in vitro and in vivo, Cell Immunol. 192 (1999) 54 – 62. [37] K.A. Hogquist, S.C. Jameson, W.R. Heath, J.L. Howard, M.J. Bevan, F.R. Carbone, T cell receptor antagonist peptides induce positive selection, Cell 76 (1994) 17 – 27. [38] M.W. Moore, F.R. Carbone, M.J. Bevan, Introduction of soluble protein into the class I pathway of antigen processing and presentation, Cell 54 (1988) 777 – 785. [39] D.H. Fremont, E.A. Stura, M. Matsumura, P.A. Peterson, I.A. Wilson, Crystal structure of an H-2Kb-ovalbumin peptide complex reveals the interplay of primary and secondary anchor positions in the major histocompatibility complex binding groove, Proc. Natl. Acad. Sci. U. S. A. 92 (1995) 2479 – 2483. [40] B. Jones, C.A. Janeway Jr., Cooperative interaction of B lymphocytes with antigen-specific helper T lymphocytes is MHC restricted, Nature 292 (1981) 547 – 549. [41] K.A. Hogquist, A.G. Grandea III, M.J. Bevan, Peptide variants reveal how antibodies recognize major histocompatibility complex class I, Eur. J. Immunol. 23 (1993) 3028 – 3036. [42] A. Porgador, J.W. Yewdell, Y. Deng, J.R. Bennink, R.N. Germain, Localization, quantitation, and in situ detection of specific peptideMHC class I complexes using a monoclonal antibody, Immunity 6 (1997) 715 – 726. [43] J.A. Bluestone, A. Kaliyaperumal, S. Jameson, S. Miller, R. Dick II, Peptide-induced changes in class I heavy chains alter allorecognition, J. Immunol. 151 (1993) 3943 – 3953. [44] K.A. Pape, A. Khoruts, A. Mondino, M.K. Jenkins, Inflammatory

[45]

[46]

[47]

[48]

[49]

[50]

[51]

[52]

[53]

[54] [55]

[56]

[57]

[58]

[59]

[60]

[61]

[62]

75

cytokines enhance the in vivo clonal expansion and differentiation of antigen-activated CD4+ T cells, J. Immunol. 159 (1997) 591 – 598. R.B. Levy, K. Ozato, J.C. Richardson, J.A. Bluestone, Molecular localization of allogeneic and self determinants recognized by bulk and clonal populations of cytotoxic T cells, J. Immunol. 134 (1985) 677 – 683. T. Boon, E. De Plaen, C. Lurquin, B. Van den Eynde, P. van der Bruggen, C. Traversari, A. Amar-Costesec, A. Van Pel, Identification of tumour rejection antigens recognized by T lymphocytes, Cancer Surv. 13 (1992) 23 – 37. Y.T. Chen, E. Stockert, S. Tsang, K.A. Coplan, L.J. Old, Immunophenotyping of melanomas for tyrosinase: implications for vaccine development, Proc. Natl. Acad. Sci. U. S. A. 92 (1995) 8125 – 8129. E. Jager, M. Ringhoffer, J. Karbach, M. Arand, F. Oesch, A. Knuth, Inverse relationship of melanocyte differentiation antigen expression in melanoma tissues and CD8+ cytotoxic-T-cell responses: evidence for immunoselection of antigen-loss variants in vivo, Int. J. Cancer 66 (1996) 470 – 476. J. Goldberg, P. Shrikant, M.F. Mescher, In vivo augmentation of tumor-specific CTL responses by class I/peptide antigen complexes on microspheres (large multivalent immunogen), J. Immunol. 170 (2003) 228 – 235. S.M. O’Herrin, J.E. Slansky, Q. Tang, M.A. Markiewicz, T.F. Gajewski, D.M. Pardoll, J.P. Schneck, J.A. Bluestone, Antigen-specific blockade of T cells in vivo using dimeric MHC peptide, J. Immunol. 167 (2001) 2555 – 2560. K.A. Pape, E.R. Kearney, A. Khoruts, A. Mondino, R. Merica, Z.M. Chen, E. Ingulli, J. White, J.G. Johnson, M.K. Jenkins, Use of adoptive transfer of T-cell-antigen-receptor-transgenic T cell for the study of T-cell activation in vivo, Immunol. Rev. 156 (1997) 67 – 78. E.R. Kearney, K.A. Pape, D.Y. Loh, M.K. Jenkins, Visualization of peptide-specific T cell immunity and peripheral tolerance induction in vivo, Immunity 1 (1994) 327 – 339. M. Bellone, D. Cantarella, P. Castiglioni, M.C. Crosti, A. Ronchetti, M. Moro, M.P. Garancini, G. Casorati, P. Dellabona, Relevance of the tumor antigen in the validation of three vaccination strategies for melanoma, J. Immunol. 165 (2000) 2651 – 2656. L. Fong, E.G. Engleman, Dendritic cells in cancer immunotherapy, Annu. Rev. Immunol. 18 (2000) 245 – 273. M.M. Soares, V. Mehta, O.J. Finn, Three different vaccines based on the 140-amino acid MUC1 peptide with seven tandemly repeated tumor-specific epitopes elicit distinct immune effector mechanisms in wild-type versus MUC1-transgenic mice with different potential for tumor rejection, J. Immunol. 166 (2001) 6555 – 6563. M. Bellone, G. Iezzi, M.A. Imro, M.P. Protti, Cancer immunotherapy: synthetic and natural peptides in the balance, Immunol. Today 20 (1999) 457 – 462. M.T. Lotze, B. Hellerstedt, L. Stolinski, T. Tueting, C. Wilson, D. Kinzler, H. Vu, J.T. Rubin, W. Storkus, H. Tahara, E. Elder, T. Whiteside, The role of interleukin-2, interleukin-12, and dendritic cells in cancer therapy, Cancer J. Sci. Am. 3 (Suppl. 1) (1997) S109 – S114. D.C. Tang, M. DeVit, S.A. Johnston, Genetic immunization is a simple method for eliciting an immune response, Nature 356 (1992) 152 – 154. H.L. Robinson, L.A. Hunt, R.G. Webster, Protection against a lethal influenza virus challenge by immunization with a haemagglutininexpressing plasmid DNA, Vaccine 11 (1993) 957 – 960. J.B. Ulmer, J.J. Donnelly, S.E. Parker, G.H. Rhodes, P.L. Felgner, V.J. Dwarki, S.H. Gromkowski, R.R. Deck, C.M. DeWitt, A. Friedman, et al., Heterologous protection against influenza by injection of DNA encoding a viral protein, Science 259 (1993) 1745 – 1749. K. Haupt, M. Roggendorf, K. Mann, The potential of DNA vaccination against tumor-associated antigens for antitumor therapy, Exp. Biol. Med. (Maywood) 227 (2002) 227 – 237. H.L. Hanson, D.L. Donermeyer, H. Ikeda, J.M. White, V. Shankaran, L.J. Old, H. Shiku, R.D. Schreiber, P.M. Allen, Eradication of

76

[63]

[64]

[65]

[66] [67]

[68]

[69] [70]

[71]

B. Carey et al. / Clinical Immunology 116 (2005) 65–76 established tumors by CD8+ T cell adoptive immunotherapy, Immunity 13 (2000) 265 – 276. G. Yang, K.E. Hellstrom, M.T. Mizuno, L. Chen, In vitro priming of tumor-reactive cytolytic T lymphocytes by combining IL-10 with B7CD28 costimulation, J. Immunol. 155 (1995) 3897 – 3903. P.W. Chen, M. Wang, V. Bronte, Y. Zhai, S.A. Rosenberg, N.P. Restifo, Therapeutic antitumor response after immunization with a recombinant adenovirus encoding a model tumor-associated antigen, J. Immunol. 156 (1996) 224 – 231. S. Sarma, Y. Guo, Y. Guilloux, C. Lee, X.F. Bai, Y. Liu, Cytotoxic T lymphocytes to an unmutated tumor rejection antigen P1A: normal development but restrained effector function in vivo, J. Exp. Med. 189 (1999) 811 – 820. T.J. Curiel, D.T. Curiel, Tumor immunotherapy: inching toward the finish line, J. Clin. Invest. 109 (2002) 311 – 312. S.A. Rosenberg, J.R. Yannelli, J.C. Yang, S.L. Topalian, D.J. Schwartzentruber, J.S. Weber, D.R. Parkinson, C.A. Seipp, J.H. Einhorn, D.E. White, Treatment of patients with metastatic melanoma with autologous tumor-infiltrating lymphocytes and interleukin 2, J. Natl. Cancer Inst. 86 (1994) 1159 – 1166. M.H. Ryan, J.A. Bristol, E. McDuffie, S.I. Abrams, Regression of extensive pulmonary metastases in mice by adoptive transfer of antigen-specific CD8(+) CTL reactive against tumor cells expressing a naturally occurring rejection epitope, J. Immunol. 167 (2001) 4286 – 4292. H. Winter, B.A. Fox, Adoptive cellular immunotherapy of cancer, Curr. Opin. Mol. Ther. 1 (1999) 89 – 97. R.L. Burke, C. Goldbeck, P. Ng, L. Stanberry, G. Ott, G. Van Nest, The influence of adjuvant on the therapeutic efficacy of a recombinant genital herpes vaccine, J. Infect. Dis. 170 (1994) 1110 – 1119. E. Caselli, P.G. Balboni, C. Incorvaia, R. Argnani, F. Parmeggiani, E. Cassai, R. Manservigi, Local and systemic inoculation of DNA or

[72] [73]

[74]

[75]

[76]

[77]

[78]

[79] [80]

protein gB1s-based vaccines induce a protective immunity against rabbit ocular HSV-1 infection, Vaccine 19 (2000) 1225 – 1231. D.I. Bernstein, L.R. Stanberry, Herpes simplex virus vaccines, Vaccine 17 (1999) 1681 – 1689. M.R. McDermott, F.L. Graham, T. Hanke, D.C. Johnson, Protection of mice against lethal challenge with herpes simplex virus by vaccination with an adenovirus vector expressing HSV glycoprotein B, Virology 169 (1989) 244 – 247. B.A. Blacklaws, S. Krishna, A.C. Minson, A.A. Nash, Immunogenicity of herpes simplex virus type 1 glycoproteins expressed in vaccinia virus recombinants, Virology 177 (1990) 727 – 736. J.E. Blaney Jr., E. Nobusawa, M.A. Brehm, R.H. Bonneau, L.M. Mylin, T.M. Fu, Y. Kawaoka, S.S. Tevethia, Immunization with a single major histocompatibility complex class I-restricted cytotoxic Tlymphocyte recognition epitope of herpes simplex virus type 2 confers protective immunity, J. Virol. 72 (1998) 9567 – 9574. P.E. Cruz, P.L. Khalil, T.D. Dryden, H.C. Chiou, P.S. Fink, S.J. Berberich, N.J. Bigley, A novel immunization method to induce cytotoxic T-lymphocyte responses (CTL) against plasmid-encoded herpes simplex virus type-1 glycoprotein D, Vaccine 17 (1999) 1091 – 1099. I.A. York, C. Roop, D.W. Andrews, S.R. Riddell, F.L. Graham, D.C. Johnson, A cytosolic herpes simplex virus protein inhibits antigen presentation to CD8+ T lymphocytes, Cell 77 (1994) 525 – 535. A.K. Abbas, A.H. Lichtman, J.S. Pober, Cellular and molecular immunology, Saunders Text and Review Series, third ed., W. B. Saunders Company, Philadelphia, 1997, p. 494. B. Meignier, Genetically engineered attenuated herpes simplex viruses, Rev. Infect. Dis. 13 (Suppl. 11) (1991) S895 – S897. S. Gurunathan, D.M. Klinman, R.A. Seder, DNA vaccines: immunology, application, and optimization, Annu. Rev. Immunol. 18 (2000) 927 – 974.