Fusion Protein Anchored Onto Tumor Cells Superantigen

Antitumor Response Elicited by a Superantigen- Transmembrane Sequence Fusion Protein Anchored onto Tumor Cells

This information is current as Jennifer L. Wahlsten, Charles D. Mills and S. Ramakrishnan of October 3, 2021. J Immunol 1998; 161:6761-6767; ; http://www.jimmunol.org/content/161/12/6761

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The Journal of Immunology is published twice each month by The American Association of Immunologists, Inc., 1451 Rockville Pike, Suite 650, Rockville, MD 20852 Copyright © 1998 by The American Association of Immunologists All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. Antitumor Response Elicited by a Superantigen- Transmembrane Sequence Fusion Protein Anchored onto Tumor Cells1

Jennifer L. Wahlsten,2* Charles D. Mills,† and S. Ramakrishnan3*

Superantigens stimulate T cells bearing certain TCR ␤-chain variable regions when bound to MHC II molecules. We investigated whether the superantigen toxic shock syndrome toxin-1 (TSST1) could induce an antitumor immune response when anchored onto MHC II-negative tumor cells. Our approach was to facilitate association of TSST1 with cell membranes by fusing its coding region to the transmembrane region (TM) sequence of the proto-oncogene c-erb-B-2. TSST1-TM was expressed in bacteria with an N-terminal histidine tag and purified using nickel-agarose affinity chromatography. Purified TSST1-TM added to cultures of several different MHC II-negative tumor cells spontaneously associated with cell membranes, as detected by flow cytometry.

Because superantigens can direct cell-mediated cytotoxicity against MHC II-positive cells, a TM fusion protein lacking the TSST1 Downloaded from

MHC II binding domain (TSST88–194-TM) was also constructed. Tumor cells precoated with TSST1-TM or TSST88–194-TM stimulated proliferation of human peripheral blood lymphocytes in vitro whereas uncoated tumor cells did not. Mice preimmunized with TSST1-TM- or TSST88–194-TM-coated tumor cells mounted a systemic response that resulted in signiﬁcant antitumor immunity as measured by regression of a parental tumor challenge. TSST1-TM and TSST88–194-TM fusion proteins represent a useful new strategy for attaching superantigens or potentially other proteins onto tumor cell surfaces without genetic manipulation. The Journal of Immunology, 1998, 161: 6761–6767. http://www.jimmunol.org/

umor cells often escape protective immune responses be- We sought to develop a strategy for passively attaching the su- cause they lack or down-regulate stimulatory surface Ags perantigen toxic shock syndrome toxin-1 (TSST1) onto tumor T (1). Introduction of immunostimulatory Ags such as cells. As a superantigen, TSST1 stimulates T cells bearing certain MHC I, MHC II, or B7-1 onto the surface of tumor cells can TCR V␤ elements when bound as an unprocessed protein outside induce antitumor immunity as demonstrated by rejection of paren- the antigenic groove of MHC II molecules (6). The subsequent tal tumor in vivo (2). Typically, these approaches involve trans- polyclonal T cell expansion results in massive cytokine release, fection of the relevant genes. However, transfection is time con- ␥ including IL-2, IFN- , and TNF (7). Local release of IL-2 or by guest on October 3, 2021 suming, and primary tumor cells often do not grow well in vitro. IFN-␥ by transfected tumor cells can elicit systemic antitumor im- Therefore, the practical application of this strategy to human tu- munity (8). Cell anchoring of a superantigen such as TSST1 would mors may be limited. place an immunostimulatory surface Ag on tumor cells capable of Alternatively, surface Ags can be introduced onto tumor cells promoting local production of these and other cytokines. The po- without transfection through fusion with a glycosyl-phosphatidyl- tential of this strategy has been demonstrated in a study where inositol (GPI)4 signal sequence (3). Puriﬁed, recombinant GPI- mice immunized with tumor cells chemically linked to the super- linked fusion proteins incubated with tumor cells can passively antigen Staphylococcal enterotoxin B developed speciﬁc antitumor re-anchor onto extracellular membranes. In vitro studies applying immunity (9). However, covalently linking proteins onto cells has this approach demonstrated that GPI-anchored, Ag-presenting the potential limitation of altering cell function, and generally does MHC I molecules effectively promoted T cell-mediated cytotoxicity (4), and GPI-anchored B7-1 (CD80) costimulated lymphocyte not allow for a single point of attachment, nor for predictable ori- proliferation (5). entation. Applying the aforementioned method of fusing TSST1 to a GPI signal sequence could result in predictable orientation and attachment. However, GPI linkage requires eukaryotic processing † Departments of *Pharmacology and Surgery, University of Minnesota, Minneapolis, (10). Therefore, as a prokaryotic protein TSST1 is not well suited MN 55455 to a GPI-signal-sequence-fusion strategy for cell membrane an- Received for publication April 14, 1998. Accepted for publication August 11, 1998. choring. We sought to develop a strategy for passive anchoring The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance that allowed TSST1 expression in a bacterial host. with 18 U.S.C. Section 1734 solely to indicate this fact. In searching for an applicable method to achieve this end, Lin et 1 J.L.W. was a Howard Hughes Medical Institute Predoctoral Fellow. C.D.M. is sup- al. (11) reported that addition of a synthetic peptide—composed of ported by National Institutes of Health PO1GM50150. a signal peptide sequence and a nuclear localization sequence—to 2 Current address: Laboratory of Immunology, National Eye Institute, National Insti- living eukaryotic cells resulted in its spontaneous cellular import tutes of Health, Building 10, Room 10N222, Bethesda, MD 20892-1857. (11). Cellular uptake of this nuclear localization sequence was pri- 3 Address correspondence and reprint requests to Dr. S. Ramakrishnan, Department of Pharmacology, University of Minnesota, 3-249 Millard Hall, 435 Delaware Street SE, marily mediated by the hydrophobic region of the signal peptide. Minneapolis, MN 55455. It has been demonstrated in Escherichia coli that hydrophobic seg- 4 Abbreviations used in this paper: GPI, glycosyl-phosphatidylinositol; TSST1, toxic ments can facilitate their own membrane insertion (12), and recent shock syndrome toxin-1; H, histidine tag; TM, transmembrane; T84, TSST1 residues studies indicate this phenomenon also applies to transmembrane 88–194; LLC, Lewis lung carcinoma; IPTG, isopropyl ␤-D-thiogalactopyranoside; Mit C, mitomycin C; SEA, Staphylococcal enterotoxin A. (TM) fragments of eukaryotic proteins (13). Since increasing

Copyright © 1998 by The American Association of Immunologists 0022-1767/98/$02.00 6762 SUPERANTIGEN-TRANSMEMBRANE CHIMERA ANCHORS ONTO TUMOR CELLS hydrophobicity will change a translocating sequence into a stop- were confirmed through NdeI/XhoI redigestion and dsDNA sequencing be- anchor sequence (14, 15), we hypothesized that fusing TSST1 to a fore transforming into host strain BL21(DE3) pLysS for expression. hydrophobic TM sequence would permit it to passively anchor Expression of HTSST1-TM and HT84-TM in E. coli onto cell membranes of living cells. Here we show that a recombinant TSST1-transmembrane region chimera (HTSST1-TM) Expression of HTSST1-TM and HT84-TM in E. coli was adapted from a described protocol (31, 32). Overnight cultures of transformed BL21(DE3) spontaneously associates with intact cells. pLysS cells were inoculated 1:40 into 1 liter of M9-ZB medium (33) sup- Superantigen-based antitumor strategies may offer therapeutic plemented with 2% glucose, 100 ␮g/ml ampicillin, and 34 ␮g/ml chlor- promise. However, because superantigens preferentially direct cy- amphenicol. Cultures were grown to an A600 of 0.8–1.0 with continuous ␤ totoxicity against MHC II-positive cells (16–18), in vivo admin- shaking at 37°C before transcription was induced with isopropyl -D-thiogalactopyranoside (IPTG, 0.4 mM). Cells were harvested after 2-h induc- istration of intact superantigens in sufficiently therapeutic amounts tion, the cell pellet was resuspended in 80 ml sonication buffer (300 mM risks unwanted cytotoxicity against normal cells. Therefore, suc- NaCl/50 mM sodium phosphate, pH 8/1% Triton X-100), and was frozen cessful employment of superantigens in tumor immunotherapy at Ϫ80°C. will likely require compromising their MHC II binding capabili- Purification of HTSST1-TM and HT84-TM ties. Crystallographic studies have demonstrated that residues within the TSST1 N-terminal domain directly interact with MHC Proteins expressed from pET 17bH have 10 sequential histidine residues at the amino terminus, allowing purification over nickel-agarose. The frozen II molecules (19), and mutation analyses of TSST1 have localized cell lysate was thawed, sonicated and clarified by centrifugation at residues critical for its superantigenic activity to the C-terminal 28,000 ϫ g for 30 min. The clarified lysate was diluted with an equal domain (20–22). When expressed as a recombinant protein, the volume of sonication buffer and loaded batchwise onto nickel agarose over TSST1 C-terminal residues 88–194 do not bind to MHC II mol- 4 h at 4°C. The resin was rinsed with sonication buffer, placed in a column Downloaded from ecules yet retain superantigenic activity (23). Therefore, a TM fu- support, and rinsed with wash buffer (300 mM NaCl/50 mM sodium phosphate, pH 8/15% glycerol). Nonspecifically bound host proteins eluted in sion protein having only the TSST1 C-terminal residues 88–194 wash buffer containing 100 mM imidazole. HTSST1-TM and HT84-TM was also constructed (HTSST88–194-TM or HT84-TM). Here we bound to the resin was eluted in wash buffer containing 300 mM imidazole show that cell-anchored HTSST1-TM and HT84-TM exhibit po- and dialysed into PBS (137 mM NaCl, 2.7 mM KCl, 4.3 mM Na2HPO4, 1.4 tent biological activity in vitro and in vivo, as measured by lym- mM KH2PO4). Purified, dialysed protein was concentrated, aliquoted, and stored at Ϫ80°C. Protein concentrations were determined using BCA assay phocyte proliferation and tumor immunization, respectively.

reagents (34). Relative protein purity was determined by densitometric http://www.jimmunol.org/ Passive anchoring of superantigen and potentially other TM fu- measurement of Coomassie brilliant blue R-250-stained 12% SDS poly- sion proteins offers a novel strategy for addition of immunostimu- acrylamide gels. latory molecules onto tumor cells. Association of HTSST1-TM with MHC II-negative tumor cells Materials and Methods Tumor cells were resuspended to 0.5–1.0 ϫ 106 cells/ml in medium con- Reagents taining 2.5% FCS. Purified HTSST1-TM was added to a final concentration of 0.33–1.67 ␮M and incubated for4hat37°C. Cells were rinsed and The prokaryotic expression vector pET17bH, with an N-terminal histidine analyzed for the presence of HTSST1 on their surface by flow cytometry. sequence, has been described (24). A TSST1 subclone was provided by Dr. In the negative control, cells not exposed to HTSST1-TM were incubated with primary and secondary Abs. Anchored HTSST1-TM was detected P. M. Schlievert (University of Minnesota, Minneapolis). Enzymes for mo- by guest on October 3, 2021 lecular cloning were purchased from New England Biolabs (Beverly, MA). with purified IgG from rabbit serum against TSST1 residues 88–194 Oligonucleotides were synthesized by Integrated DNA Technologies (Cor- (␣T84), added at a 1:300 dilution with 3% normal goat serum. Bound ␣T84 alville, IA). FITC-labeled goat-anti-rabbit IgG Abs were obtained from was detected with FITC goat anti-rabbit IgG added at a 1:200 dilution. Sigma (St. Louis, MO). Murine cell lines EL4 and Lewis lung carcinoma Cells were gated for viability using propidium iodide and 2–10 ϫ 103 (LLC) (C57BL/6, H-2b) and the plasmid encoding c-erb-B-2 were pur- events were measured for positive fluorescence by flow cytometry. chased from the American Type Culture Collection (Manassas, VA). Raji The presence of HTSST1-TM on the human ovarian carcinoma cell MHC II-negative mutant RJ2.2.5 was obtained from Dr. J. M. Boss (Emory line, MA 148 (35), was also visualized by in situ immunohistochemistry. University, Atlanta, GA) with permission of Dr. R. Accolla (Advanced MA 148 cells were seeded at low density onto glass coverslips and grown Biotechnology Center, Genova, Italy). The murine P815 mastocytoma for 24–48 h. The cells were then incubated for4hat37°C in medium (DBA/2, H-2d) used has been described (25–27). Female C57BL/6 ϫ containing 1 ␮M HTSST1-TM and 2.5% serum. Cells were rinsed with b/d DBA/2 (B6D2F1, H-2 ) 8-wk-old mice were purchased from Taconic PBS and fixed for 10 min at room temperature (4% paraformaldehyde, 2% Farms (Germantown, NY) and housed within University of Minnesota an- sucrose in PBS). Detection of HTSST1-TM was done using the Abs de- imal facilities. The P815 tumor grows progressively and has been shown to scribed above. Coverslips were mounted onto glass slides. Images were ϫ generate a tumor-specific response in B6D2F1 mice as well as in the pa- captured with a 10 neofluor objective on a Nikon Diaphot 300 inverted rental strain (25–27). microscope connected to a Photometrics PXL cooled CCD camera (Tuc- son, AZ) using the computer program IPLab Spectrum (Signal Analytics, Construction and subcloning of the TSST1-TM and T84-TM Vienna, VA). fusion sequences into pET 17bH Biological activity of HTSST1-TM and HT84-TM in vitro

The coding sequences for TSST1 or TSST88–194 were fused to the TM- encoding sequence of c-erb-B-2 using splice overlap extension PCR (28) The in vitro biological activity of HTSST1-TM- or HT84-TM-coated tu- and the following primers, with restriction sites in bold and an underlined mor cells was measured using a lymphocyte proliferation assay. PBL were stop codon: primer 1, 5Ј-CCCCATATGTCTACAAACGATAATATAAA isolated from the blood of healthy donors over a Histopaque 1077 gradient ϫ 5 GGAT-3Ј; primer 2, 5Ј-TCTCTGCTCGGCACTAGTATTAATTTCTGC (Sigma) and aliquoted to 2 10 cells/well in 96-well U-bottom plates. 3Ј; primer 3, 5Ј-GCAGAAATTAATACTAGTGCCGAGCAGAGA-3Ј; The PBLs were incubated in 0.2 ml RPMI 1640 medium containing 5% primer 4, 5Ј-CCCCTCGAGTTACATCGTGTACTTCCG-3Ј; primer 5, FCS and irradiated (10,000 rad) coated or uncoated MA 148 cells at a 1:1 Ј Ј or 1:2 tumor cell to PBL ratio. MA 148 cells were coated with either 1.67 5 -CCCCATATGGTTACAAATACTGAA-3 . ␮ The TSST1 coding sequences were amplified from MNT subclone 6101 M of HTSST1-TM or HT84-TM, or serial 5-fold dilutions of HTSST1- (29) using primers 1 and 2 or 5 and 2. The DNA sequence corresponding TM. After 72-h incubation (37°C, 5% CO2), cells were labeled with [3H]thymidine (1 ␮Ci/well) for 18 h before harvesting. Incorporation of to amino acid residues 644–687 of c-erb-B-2, inclusive of the TM region, 3 was amplified from plasmid clone pCER204 (30) using primers 3 and 4. [ H]thymidine was determined using a liquid-scintillation counter. The amplified fragments corresponding to TSST1 (610 bp), T84 (320 bp), Biological activity of HTSST1-TM and HT84-TM in vivo and c-erb-B-2 (160 bp) were purified and used as templates in subsequent PCRs. The TSST1-TM fusion sequences were PCR-amplified from the The in vivo biological activity of HTSST1-TM- or HT84-TM-coated tumor TSST1 or T84 and c-erb-B-2 fragments using primers 1 and 4 or 5 and 4, cells was determined using an established immunization protocol (25–27). respectively. The amplified fragments (740 bp and 450 bp) were digested Five mice were in each treatment group. P815 cells (1 ϫ 106 cells/ml) were with NdeI and XhoI and ligated into predigested pET 17bH. Positive clones incubated in serum-free medium alone or with HTSST1-TM (0.17 or 1.67 The Journal of Immunology 6763

␮M) or HT84-TM (0.03, 0.17, or 0.85 ␮M) as described above. Tumor cells were then rinsed, resuspended at 1 ϫ 107 cells/ml, and treated with 50 ␮g/ml mitomycin C (Mit C) for1hat37°C. Mice were immunized s.c. in the foot with superantigen-coated or control tumor cells (1 ϫ 107 cells in 50 ␮l/mouse). At 21 days postimmunization, antitumor immunity was determined by challenging mice intradermally in the axillary region with 5 ϫ 105 or 1 ϫ 106 viable P815 tumor cells. Previous studies using this tumor immunization model have shown that the primary P815 tumor-specific CTL response disappears by about 14–17 days, so that by 21 days a state of long-term memory immunity is present. Also, by waiting 21 days we reduce the possible contribution of nonspecific effects due to the superantigen. Bisecting tumor diameters were measured with calipers every 2–3 days. Mice were sacrificed when tumor diameters exceeded 15 mm. All control mice developed progressively growing tumors. Some mice in the treatment groups did not, and of the tumors that did develop, some pro- gressed and others regressed. Therefore, significant differences between control and test groups were determined using the nonparametric Wilcoxon Rank Sum test. Results Cloning and expression of HTSST1-TM and HT84-TM The chimeric TSST1 or T84/c-erb-B-2 TM region DNA sequences Downloaded from were created by splice overlap extension (28) and subcloned into pET 17bH as described in Materials and Methods and depicted in Fig. 1A. Control of the chromosomally located T7 polymerase gene in E. coli host strain BL21(DE3) is not completely stringent, resulting in basal expression of proteins directed by the T7 pro- moter (33). Considerable levels of HTSST1 were expressed in BL21(DE3) without induction of T7 polymerase by IPTG (32). http://www.jimmunol.org/ HTSST1-TM and HT84-TM are toxic to bacteria which express them, but can be successfully over-expressed if protein production is stringently repressed during growth. Expression of HTSST1-TM and HT84-TM was achieved when transforming the TSST1-TM/ pET 17bH plasmids into E. coli host strain BL21(DE3) pLysS and growing cultures in 2%, rather than 0.4%, glucose (31). Successful expression of TSST1-TM and T84-TM under these more stringent

growth conditions is shown on a Coomassie brilliant blue-stained by guest on October 3, 2021 12% SDS-polyacrylamide gel of whole bacterial lysates from induced and uninduced cultures (Fig. 1B). Only IPTG-induced cultures over-expressed recombinant protein. FIGURE 1. A, Schematic of the HTSST1-TM and HT84-TM proteins, Purification of HTSST1-TM and HT84-TM with sequences created by splice overlap PCR. Arrows indicate relative HTSST1-TM and HT84-TM were extracted from bacterial cell positions of the primers. Boxes are not proportional. Subcloning of the membranes using the nonionic detergent Triton X-100 (1%) be- fusion sequences into pET 17bH results in a N-terminal histidine sequence. B and C, Representative 12% SDS-PAGE analyses showing expression and cause refolded preparations extracted using denaturing agents such purification of HTSST1-TM and HT84-TM. B, Expression of HTSST1, as urea or guanidine-HCl did not induce proliferation. Protein in HTSST1-TM, and HT84-TM in host BL21(DE3) pLysS. Pelleted 1 ml the clarified bacterial lysate loaded more efficiently onto the nick- aliquots of 3 ml minicultures were directly resuspended in 0.5 ml sample el-agarose resin when incubated batchwise. The resin was then buffer (50 mM Tris-HCl (pH 6.8), 100 mM DTT, 2% SDS, 0.1% bromo- thoroughly rinsed while on an immobilized support with a wash phenol blue, 10% glycerol); 20 ␮l were loaded per well. Lane 1, uninduced buffer containing glycerol. A rinse containing a comparatively HTSST1; lane 2, induced HTSST1; lane 3, uninduced HTSST1-TM; lane high concentration of imidazole (100 mM) was necessary to elute 4, induced HTSST1-TM; lane 5, uninduced HT84-TM; lane 6, induced host proteins nonspecifically associating with either the TM fusion HT84-TM. C, Purified HTSST1-TM (5 ␮g loaded) and HT84-TM (2 ␮g proteins or the resin. Upon addition of 300 mM imidazole, loaded). HTSST1-TM or HT84-TM eluted between 4 and 20 ml. Positive fractions were pooled and dialysed against PBS. Fig. 1C depicts a representative Coomassie brilliant blue-stained 12% SDS-poly- lymphoma, Raji (36). Murine cell lines EL4, LLC, P815 and the acrylamide gel showing purified HTSST1-TM and HT84-TM. human ovarian carcinoma MA 148 are all MHC class II-negative, This one-step purification strategy yielded per liter of bacterial even after exposure to IFN-␥ (data not shown; Ref. 37). culture 2 mg of HTSST-TM or 0.2 mg of HT84-TM with an av- Spontaneous anchoring of HTSST1-TM onto the different tumor erage relative purity of 88%. cell lines was determined by flow cytometry. The cells were incubated with HTSST1-TM or HTSST1 at 37°C, rinsed of unbound HTSST1-TM associates with MHC II-negative tumor cells protein, and immediately prepared for flow cytometry. As shown Superantigens normally bind MHC class II molecules on cells (6). in Fig. 2, HTSST1-TM spontaneously anchored onto all tumor cell The ability of HTSST1-TM to associate with cells through the TM lines tested. This association requires the TM region, because re- region but not by MHC II binding was evaluated on four different combinant native HTSST1 did not bind to RJ2.2.5, MA 148, or MHC II-negative tumor cell lines: MA 148, RJ2.2.5, LLC, and LLC cells (Fig. 2). The cell lines tolerated serum deprivation vari- EL4. RJ2.2.5 is a MHC II-negative mutant of the human B cell ably, regardless of exposure to HTSST1-TM. Addition of serum 6764 SUPERANTIGEN-TRANSMEMBRANE CHIMERA ANCHORS ONTO TUMOR CELLS

FIGURE 2. HTSST1-TM spontaneously associates with MHC II-negative tumor cell lines. Coated tumor cells MA 148 (7500 events), RJ2.2.5 Downloaded from (10,000 events), LLC (7500 events), and EL4 (2000 events) were analyzed FIGURE 4. HTSST1-TM- and HT84-TM-coated MA 148 cells stimu- ␣ by ﬂow cytometry using puriﬁed T84 Ig, and FITC goat anti-rabbit Ig. late proliferation of human PBLs. A, MA 148 cells were coated with 1.67 ⅐⅐⅐ Fluorescence was measured on cells exposed to serum-free medium ( ), ␮M HTSST1-TM or HT84-TM, irradiated, and added at a 1:1 tumor/lym- ␮ 0.75 M HTSST1(---), or HTSST1-TM (——). HTSST1 binding to EL4 phocyte (T:L) ratio to 2 ϫ 105 PBLs/well in a 96-well plate. B, Coated MA cells was not tested. 148 cells were preincubated with 5-fold serial dilutions of HTSST1-TM and added at a 1:2 T:L ratio. Values on the y-axis represent the average

cpm Ϯ SD of triplicate cultures from two independent experiments. Back- http://www.jimmunol.org/ concentrations up to 2.5% did not interfere with HTSST1-TM as- p Ͻ 0.004 vs ,ء .ground values of lymphocytes alone have been subtracted sociation and improved viability. MA 148 cells prepared for flow uncoated MA 148, two-tailed Student’s t test. cytometry 22 h after incubation with HTSST1-TM retained a fluorescence intensity at 25% the level of treated cells that were analyzed directly (data not shown). This is most likely due to inter- HTSST1-TM- or HT84-TM-stimulated lymphocyte proliferation nalization or shedding of HTSST1 into the medium, because (Fig. 4A). To determine whether cells coated with smaller concen- preventing cell division with Mit C did not change this result. trations of protein were biologically active, MA 148 cells were HTSST1-TM did not significantly associate with cells when incu- coated with 5-fold serial dilutions of HTSST1-TM before being

bated at 4°C (data not shown). To determine the distribution of cultured with PBLs (Fig. 4B). Preincubating MA 148 cells with as by guest on October 3, 2021 HTSST1-TM association among coated cells and retention of cell little as 1 nM HTSST1-TM induced significant PBL proliferation integrity after incubation, anchored HTSST1-TM on MA 148 was vs uncoated MA 148 cells ( p Ͻ 0.004, Fig. 4B). These results also visualized by indirect immunofluorescence (Fig. 3). show that membrane bound HTSST1-TM and HT84-TM can induce lymphocyte proliferation in vitro. MA 148 cells coated with HTSST1-TM or HT84-TM stimulate lymphocyte proliferation Immunization with HTSST1-TM- or HT84-TM-coated P815 cells induces antitumor immunity The in vitro biological activity of HTSST1-TM- or HT84-TM- coated MA 148 cells was determined by lymphocyte proliferation The in vivo biological activity of HTSST1-TM- or HT84-TM- assays. Fig. 4A shows the results of an experiment where MA 148 coated murine P815 cells was determined using an established im- cells were coated with 1.67 ␮M of HTSST1-TM or HT84-TM munization protocol that measures a secondary response against before being cultured with PBLs. MA 148 cells coated with either parental tumor challenge (25–27). Mice (five per group) were immunized in the footpad with Mit C-treated P815 cells or P815 cells precoated with varying concentrations of HTSST1-TM or HT84- TM. We used P815 tumor cells for in vivo experiments because it is a well-characterized immunogenic tumor (38). Twenty-one days postimmunization, growth of parental tumor challenge was measured at a distant site. Mice preimmunized with either HTSST1- TM- or HT84-TM-coated P815 tumor cells exhibited significant antitumor immunity. We show in Fig. 5A that preimmunization of mice with P815 cells incubated with either 1.67 ␮M or 0.17 ␮M HTSST1-TM displayed antitumor immunity. Mice preimunized with P815 cells coated with 0.17 ␮M HTSST1-TM elicited a significant antitumor response in comparison to mice preimmunized with uncoated P815 tumor cells ( p Յ 0.03). The average diameters for this group reflect a reduced tumor incidence (three of five FIGURE 3. Visualizing HTSST1-TM on MA 148 cells by indirect immunofluorescence. MA 148 cells attached to glass coverslips were incu- mice), delayed appearance of tumors that did develop, and partial bated in medium containing 2.5% serum without (A and B) or with 1.0 ␮M regression of one tumor that eventually completely regressed (Fig. HTSST1-TM (C and D) for 4 h. Cells were rinsed, fixed, and incubated first 5B). Mice preimmunized with P815 cells incubated with 1.67 ␮M with purified ␣T84 Ig, then with FITC goat anti-rabbit Ig. A and C, Phase- of HTSST1-TM all initially developed tumor; however, the aver- contrast exposures; B and D, fluorescence exposures. Bar ϭ 30 ␮m. age value presented for this group reflects the complete regression The Journal of Immunology 6765 Downloaded from FIGURE 5. Mice immunized with HTSST1-TM- or HT84-TM-coated P815 cells mount a systemic antitumor response against challenge with parental tumor. Five mice were in each treatment group. P815 cells (1 ϫ 106/ml) were incubated with HTSST1-TM (1.67 or 0.17 ␮M), or HT84-TM (0.85, 0.17, ␮ ϫ 7 or 0.03 M) as described in Materials and Methods. B6D2F1 mice were then immunized with 1 10 HTSST1-TM- (A) or HT84-TM- (C) coated or uncoated Mit C-treated P815 cells and challenged 21 days later by axillary intradermal injection with 5 ϫ 105 (A)or1ϫ 106 (C) parental P815. B and D, P815 tumor growth rejection by the five individual mice treated with P815 cells coated with either 0.17 ␮M HTSST1-TM or 0.85 ␮M HT84-TM, .p Յ 0.03 vs P815 immunized control, Wilcoxon Rank Sum test ,ء .respectively. Values on the y-axis represent the average bisecting tumor diameters http://www.jimmunol.org/

of one tumor and the partial regression of two others (Fig. 5A). Be- cally active in vitro, as shown by the ability of coated MA 148 cause the higher dose of HTSST1-TM was less effective and because cells to elicit proliferation of PBLs. we challenged at a site distant from immunization after 21 days, the TSST1 is a powerful immunostimulant, and tumor cells coated antitumor immunity observed was very likely tumor speciﬁc. The with concentrations below the levels of detection still induced a mice with tumors that completely regressed (one from each test biological response. Incubating concentrations of at least 25 nM group) were rechallenged with 1 ϫ 106 parental P815 cells 87 days HTSST1-TM could be detected on MA 148 cells by ﬂow cytom-

postimmunization and neither developed tumor (data not shown). etry (data not shown). Yet coated MA 148 cells still stimulated by guest on October 3, 2021 We show in Fig. 5C that HT84-TM also increased the immu- significant PBL proliferation when incubated with HTSST1-TM nogenicity of P815 tumor cells. Mice preimmunized with P815 concentrations 25-fold less than this. Incubating MA 148 cells with cells incubated with 0.85 ␮M HT84-TM exhibited significant an- as little as 1 nM HTSST1-TM elicited a significant proliferative titumor immunity in comparison to mice preimmunized with un- response in vitro (Fig. 4B). This result is not surprising when con- coated P815 tumor cells ( p Յ 0.02). Of the five mice in this group, sidering that soluble TSST1 concentrations as low as 2 pM will one did not develop tumor and two developed tumors that com- induce significant proliferation of human peripheral blood mono- pletely regressed (Fig. 5D). All mice preimmunized with P815 nuclear cells in vitro (39). cells incubated with 0.17 ␮M HT84-TM initially developed tumor, Coating tumor cells with smaller concentrations of but two tumors completely regressed and one partially regressed HTSST1-TM is not only more desirable for minimizing toxicity (Fig. 5C). When comparing the antitumor responses elicited by with the potential therapeutic application of this strategy but also equimolar concentrations of HT84-TM and HTSST1-TM (0.17 resulted in better systemic antitumor immunity. Immunization with ␮ ␮M of HTSST1-TM or HT84-TM), HTSST1-TM-coated cells P815 tumor cells coated with 0.17 M HTSST1-TM significantly were more potent immunogens, correlating with the in vitro pro- inhibited subsequent onset and growth of a parental tumor chal- ␮ liferation experiments (Fig. 4A). Together, these results suggest lenge, whereas P815 cells incubated with 1.67 M was less effec- that HTSST1-TM- or HT84-TM-coated P815 tumor cells can stim- tive. It is not totally unexpected that P815 cells coated with higher ulate systemic antitumor immunity. Optimizing the immunization concentrations of HTSST1-TM were less effective immunogens. protocol should further improve the antitumor immunity induced Miethke et al. (40) have demonstrated a dose-response relationship by HTSST1-TM- or HT84-TM- anchored tumor cells. between superantigen-induced T cell stimulation or inhibition in vivo, where increasing superantigen doses resulted in anergy and deletion. The ability of high doses of superantigen to cause T cell Discussion anergy or deletion in vivo has also been documented by others (41–44). Thus, P815 cells coated with the highest concentration of In this study, we describe a novel and effective method for HTSST1-TM (1.67 ␮M) may have induced a diminished antitumor incorporating the superantigen TSST1, and perhaps other pro- response because of T cell anergy or deletion. A possible mecha- teins, onto the surface of tumor cells. Fusing a TM sequence nism for this effect might be the ability of TSST1 to directly stim- onto TSST1 resulted in its spontaneous association with four ulate macrophages through MHC II engagement (45–47). Exces- different tumor cell lines. Because the hydrophobic TM se- sive stimulation of macrophages has been shown to have quence mediates this passive anchoring, HTSST1-TM and immunosuppressive consequences (48), in part mediated by nitric HT84-TM should associate with membranes of most cells or oxide and TNF-␣, that can interfere with a productive antitumor tumors. Anchored HTSST1-TM and HT84-TM were biologi- response (26, 49). 6766 SUPERANTIGEN-TRANSMEMBRANE CHIMERA ANCHORS ONTO TUMOR CELLS

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