Cancer Gene Therapy (2002) 9, 946 – 950 D 2002 Nature Publishing Group All rights reserved 0929-1903/02 $25.00 www.nature.com/cgt

MHC class I and class II presentation of tumor in retrovirally and adenovirally transduced dendritic cells J Mark Sloan,y Michael H Kershaw, Christopher E Touloukian, Rejean Lapointe, Paul F Robbins, Nicholas P Restifo, and Patrick Hwu Center for Cancer Research, National Cancer Institute, National Institutes of Health, Bethesda, MD 20892, USA.

The unique antigen-presenting capabilities of dendritic cells (DCs) make them an attractive means with which to initiate an antitumor . Using DCs transduced with tumor for immunotherapy has several theoretical advantages over -pulsed DCs including the possibility that transduced DCs are capable of presenting on both class I and class II MHC molecules. To test this theory, we inserted the human tumor antigen gp100 into mouse DCs transgenic for HLA-DRb1*0401 using either adenoviral vector or a VSV-G pseudotyped retroviral vector. DCs transduced with tumor antigen were able to be recognized by both a murine CD8 + T-cell clone and a murine CD4 + T-cell line in a release assay, thereby demonstrating presentation of both MHC class I and class II gp100 epitopes. This study describes the simultaneous presentation of a tumor-associated antigen to both CD4 + and CD8 + T cells and lends support to the use of gene-modified DCs as a means to initiate both CD4 + and CD8 + antitumor responses. Cancer Gene Therapy (2002) 9, 946–950 doi:10.1038/sj.cgt.7700509 Keywords: adenovirus; retrovirus; gp100; melanoma;

umor-antigen–reactive T cells have been detected in the CD4 + T cells play crucial roles in antitumor responses it is Tcirculation of patients with cancer.1 However, despite therefore advantageous to develop a method whereby DCs this, tumors continue to progress in most patients, potentially can simultaneously present MHC class I and II tumor antigen because tumor-reactive T cells are in a nonresponsive state epitopes. and low in number and may need to be activated to achieve Genetic modification of DCs has been shown to result in an antitumor response. Many different methods are being presentation of a number of MHC class I and II restricted attempted for the activation of T cells including vaccines epitopes to T cells including those from beta-galactosidase,8 composed of from tumor-associated antigens2 but direct presentation by DCs of tumor-associated antigen and viral3 or DNA vectors4 encoding these antigens. An via both MHC class I and II to tumor-specific T cells is less alternative strategy involves the use of dendritic cells (DCs) well characterized. that constitute a particularly attractive adjuvant for T-cell We have previously developed methods for gene- stimulation because they play a pivotal role in the initiation modifying DCs,8 and in this study we used these methods of immunity.5 DCs are able to stimulate both CD8 + and to insert the human tumor antigen gp100 into DCs. Human CD4 + subsets of T cells, both of which are important in gp100 has well-defined class I and class II epitopes generating a robust antitumor response.5 restricted to H-2Db and HLA-DRb1*0401.9,10 Using T- DCs loaded with tumor antigens have been shown to cell lines specific for these two epitopes, we were able to inhibit tumor growth in several mouse models.6,7 Methods demonstrate that retrovirally and adenovirally transduced used to load tumor antigen in these studies include pulsing DCs are able to simultaneously present epitopes to CD4 + DCs with defined peptide epitopes or with tumor lysates. and CD8 + cells. These methods primarily result in antigen presentation on MHC class I or class II molecules that stimulate either CD8 + or CD4 + , respectively. However, because both CD8 + and Materials and methods Animals and peptides Murine class II–deficient, DR4-IE transgenic mice11 ex- Received June 12, 2002. Address correspondence and reprint requests to: Dr Patrick Hwu, Room press chimeric class II molecules consisting of the antigen- 2B42 Building 10, National Institutes of Health, Bethesda, MD 20892, binding domains from HLA-DRa and HLA-DRb1*0401 d USA. E-mail: [email protected] molecules and the remaining domains from the IE -a2 d II yJMS is a Howard Hughes Medical Institute-National Institutes of and IE -b2 chains . Animals were obtained from Taconic Health research scholar. (Germantown, NY) and maintained in accordance with Antigen presentation by transduced dendritic cells JM Sloan et al 947 institutional standards. The following peptides were synthe- entiation into DCs. After replating the DCs on day 6, the sized using standard fluorenylmethoxycarbonyl (F-moc) adenovirus was added at a multiplicity of infection of 500:1 chemistry. Mouse gp10025 – 33: EGSRNQDWL (restricted by and allowed to incubate 24 hours at 378C, 5% CO2. H-2Db), human gp10025 – 33: KVPRNQDWL (restricted by + + H-2Db), and human gp10044 –59: RQLYPEWTEAQRL Generation of murine CD8 and murine CD4 T-cell lines (restricted by HLA-DRb1*0401). The molecular masses Generation of the CD8 + T-cell clone specific for the H-2Db of peptides were verified by laser desorption mass restricted of gp100 (gp10025 –33 ) used in this spectrometry (Biosynthesis, Lewisville, TX). Purity was experiment has been described in detail elsewhere.9 Briefly, also determined in this manner and found to be >99%. CD8 + T cells were derived from splenocytes of mice immunized by gene gun with human gp100 DNA. This line Production and retroviral transduction of DC was thereafter restimulated with 1 M mouse gp10025 – 33 DCs were produced from bone marrow of DR4-IE mice as peptide. These cells are able to recognize both human and b follows. Bone marrow was expelled from the long bones of murine gp10025 – 33 epitopes presented on D molecules, DR4-IE mice. Erythrocytes were depleted in ACK lysing despite a three difference between the murine and buffer (Biofluids, Rockville, MD). Cells expressing B220, human epitope. All T cells were used between 5 and 10 days I-Ab (MHC class II), CD8, and CD4 were removed using after restimulation. Generation of the CD4 + T-cell line used hybridoma supernatant from TIB-146, TIB-229, TIB-150, in this experiment has been described in detail elsewhere.10 TIB-207 (ATCC, Manassas, VA) (0.2 mL of each super- Briefly, they were derived from DR4-IE transgenic mice that natant per 107 cells) followed by incubation with rabbit were originally immunized with hgp100 protein. Cells were complement (10 mL per 108 cells) (Cedarlane, Ontario, subsequently stimulated with irradiated syngeneic DR4-IE Canada). The remaining cells were plated at a concentration splenocytes pulsed with hgp10044 – 59. of 7Â105 cells/mL in six well plates, and cultured for 6 days in DC medium (RPMI supplemented with 5% heat- IFN-g release assay inactivated FCS (both from Gibco BRL), 2 mM glutamine After the DC harvest on day 7, 1Â105 viable T cells and (Biofluids), 100 U/mL penicillin and 100 g/mL strepto- 5 À 5 1Â10 DCs were incubated in duplicate for 12 hours in mycin (Biofluids), and 5Â10 M 2-mercaptoethanol 96-well plates. The murine IFN-g concentration in the (Sigma, St. Louis, MO). Recombinant murine GM-CSF supernatant was determined by ELISA, using commercially was added to a final concentration of 20 ng/mL (Peprotech, available reagents according to the manufacturer’s instruc- Rocky Hill, NJ) and recombinant murine IL-4 (Peprotech) tions (Endogen, Rockford, IL). Peptide-pulsed DCs were added to a final concentration of 100 ng/mL. were also used as targets in some assays, in which case replenished on days 2, 4, and 6. On day 6, nonadherent cells 6 nontransduced DCs were pulsed with 1 M peptide for were collected and replated at 1Â10 cells/mL in a six-well MHC class I–restricted epitopes or 10 M peptide for MHC plate. On day 7, the nonadherent cells were collected and class II–restricted epitopes. washed three times in PBS before use. The retroviral plas- mids pCLNCGFP and pCLNChgp100 contain the gene encoding GFP or gp100 driven by a CMV promoter em- Results bedded within a Moloney Murine Leukemia Virus backbone. DCs derived from DR4-IE transgenic mice can be The pCLNC plasmid and a plasmid encoding the VSV-G efficiently transduced using adenovirus or VSV-G envelope protein were cotransfected into 293 cells consti- pseudotyped retrovirus tutively expressing the gag and pol proteins.12 Serial super- natant harvests were performed on days 2, 3, 4, and 5 To determine the transduction efficiency of HLA- posttransfection of the 293GP cells, passed through a 0.45- DRb1*0401 transgenic DCs with retroviral and adenoviral m filter (Millipore, Bedford, MA), and frozen. Super- vectors, GFP-transduced DCs derived from DR4-IE mice natants were thawed on the day of transduction and were analyzed by flow cytometry. Both adenovirally and concentrated in an ultracentrifuge (50,000Âg, 1.5 hours, retrovirally transduced cells expressed GFP (Fig 1, a and b). 48C). Because retroviral transduction requires proliferating However, GFP was expressed in a greater percentage of cells, bone marrow cells were exposed to retroviral super- retrovirally transduced DCs (50%) compared to adenovir- natants during the proliferative phase of culture. One day ally transduced DCs (20%). Expression levels were also after bone marrow harvest, the concentrated viral supernatant higher in DCs following transduction with retrovirus when was added to the DCs along with 8 g/mL polybrene compared to adenovirus. Retrovirally and adenovirally (Sigma, St. Louis, MO) and 10 mM HEPES (Biofluids). transduced cells were also strongly positive for HLA-DR Cells were then spun on a tabletop centrifuge for 1 hour expression (Fig 1, c and d). Both types of GFP-transduced (1000Âg,25–308C). cells also displayed cell surface markers similar to non- transduced DCs. Specifically, the DCs were strongly positive Adenoviral transduction of DCs for CD11c, B7.1, and B7.2 expression (data not shown).

The recombinant adenoviral vectors Ad.GFP and Ad.hgp100 + were a gift from Bruce Roberts (Genzyme, Framingham, A murine CD8 T-cell clone specific for hgp10025 – 33 MA) and are described previously.13 Because adenoviral specifically recognizes hgp100-transduced DCs transduction is transient, bone marrow cells were exposed to To determine if the transduced DCs were able to process and recombinant adenovirus following proliferation and differ- present a class I epitope encoded by the transgene, a murine

Cancer Gene Therapy Antigen presentation by transduced dendritic cells JM Sloan et al 948 300

200

100 gamma (ng/ml) 0 Ad GFP Ad Retro Retro DC + DC + gp100 GFP gp100 control gp100 peptide (44-59) Figure 3 Transduced DCs present antigen on MHC class II. DCs were transduced with either adenovirus or retrovirus encoding the tumor antigen gp100 or GFP. CD4 + T cells specific for human

gp10044 – 59 (restricted by HLA-DRb1*0401) were able to specifically recognize gp100-transduced DCs but not GFP-transduced DCs as seen by IFN-g following overnight coculture. Transduction of gp100 using retrovirus produced DCs that were recognized at a Figure 1 Transduced DCs express GFP and MHC class II. DCs were higher level than DCs transduced with adenovirus. T cells also transduced with either retroviral CLNC-GFP (a,c) or adenoviral Ad- secreted IFN-g in response to DCs pulsed with human gp10044 – 59. GFP (b,d) and analyzed by flow cytometry. GFP expression was clearly seen in both retrovirally (a) and adenovirally (b) GFP- transduced DCs (thin line) compared to gp100-transduced DCs (thick line). However, lower levels of GFP were detected in ade- novirally-transduced cells compared to retrovirally-transduced cells. transduced DCs. This clone was able to specifically recog- High levels of MHC class II were also demonstrated in retrovirus- nize the gp100-transduced DCs above GFP-transduced transduced (c) and adenovirus-transduced (d) DCs when stained controls for both adenovirally and retrovirally transduced with specific for DRb1*0401 (thin line) compared to isotype cells (Fig 2). Recognition of adenovirally transduced DCs control (thick line). was lower than retrovirally transduced DCs, which may be due to lower expression levels of gp100 in adenovirally CD8 + T-cell clone specific to the H-2Db epitope of gp100 transduced cells. Numbers given are representative of sev- (hgp10025 – 33 ) was used in a cytokine release assay with the eral experiments. This indicates that the transduced DCs efficiently presented transgene-encoded epitopes along the presented endogenous MHC class I pathway. 250

+ 200 A murine CD4 T-cell line specific for hgp10044 – 59 specifically recognizes hgp100-transduced DCs 150 DCs identical to those used in the CD8 + assay were used in a cytokine release assay with CD4 + T cells recognizing the 100 HLA-DRb1*0401–restricted hgp10044 – 59 epitope. These CD4 + T cells were able to specifically recognize the gp100- 50 transduced DCs above GFP-transduced controls for both

Interferon gamma (ng/ml) 0 adenovirally and retrovirally transduced DCs (Fig 3). This indicates that transduced DCs were also able to process and Ad GFP Ad Retro Retro DC + DC + present a class II epitope from an endogenous antigen. gp100 GFP gp100 control gp100 Activating the transduced DCs with CD40L had little peptide (25-33) additional effect on either class I or class II presentation Figure 2 Transduced DCs present antigen on MHC class I. DCs (data not shown). transduced with adenovirus or retrovirus encoding GFP or human gp100 were cocultured with a mouse T-cell line specific for human b gp10025 – 33 (restricted by H-2D ). In this representative experiment, specific recognition of gp100-transduced DCs was evident from the Discussion higher levels of IFN-g secretion in response to gp100-transduced cells when compared to GFP-transduced cells. DCs transduced with gp100 using retrovirus were recognized more strongly than those In previous work we developed methods of transducing DCs transduced with adenovirus. Interestingly, T cells secreted IFN-g in and demonstrated that injection of DCs transduced with a response to DCs pulsed with gp100 peptide at a lower level than that model antigen gene encoding beta-galactosidase into mice seen in response to retrovirally transduced DC. could inhibit the growth of established lung metastases

Cancer Gene Therapy Antigen presentation by transduced dendritic cells JM Sloan et al 949 expressing beta-galactosidase.8 Certain endogenous anti- 2. Rosenberg SA, Yang JC, Schwartzentruber DJ, et al. Immuno- gens have previously been shown to be efficiently presented logic and therapeutic evaluation of a synthetic peptide vaccine on MHC class II molecules,14 and several groups have for the treatment of patients with metastatic melanoma. Nat observed that immunizations with transduced DCs require Med. 1998;4:321–327. CD4 + T-cell help for optimal antitumor effects;15 however, 3. Rosenberg SA, Zhai Y, Yang JC, et al. Immunizing patients + with metastatic melanoma using recombinant adenoviruses presentation of endogenous tumor antigen to CD4 T cells encoding MART-1 or gp100 melanoma antigens. J Natl by transduced DCs is less well documented. Cancer Inst. 1998;90:1894–1900. Peptides bound to MHC class I molecules come pre- 4. Restifo NP, Ying H, Hwang L, Leitner WW. The promise of dominantly from proteins degraded in the cytoplasm, where- nucleic acid vaccines. Gene Ther. 2000;7:89–92. as peptides bound to MHC class II molecules are mostly 5. Banchereau J, Steinman RM. Dendritic cells and the control of derived from exogenous or intravesicular sources.16 Re- immunity. Nature. 1998;392:245–252. cently a more complicated picture of antigen processing is 6. Zitvogel L, Mayordomo JI, Tjandrawan T, et al. Thera- emerging, in which specialized antigen-presenting cells py of murine tumors with tumor peptide-pulsed dendrit- in particular are able to sample proteins from either ic cells: dependence on T cells, B7 costimulation, and T compartment for presentation on both class I and class II helper cell 1-associated cytokines. J Exp Med. 1996;183:87– 17 97. molecules. 7. Paglia P, Chiodoni C, Rodolfo M, Colombo MP. Murine Here we demonstrate that adenovirally and retrovirally dendritic cells loaded in vitro with soluble protein prime transduced DCs are capable of presenting class I and class II cytotoxic T against tumor antigen in vivo. J Exp epitopes of the differentiation antigen, gp100. Med. 1996;183:317–322. Future studies will be important to generalize these findings 8. Specht JM, Wang G, Do MT, et al. Dendritic cells retrovirally to other antigens, as well as comparing these transduction transduced with a model antigen gene are therapeutically methods using in vivo tumor models. Like other melanosome effective against established pulmonary metastases. J Exp Med. membrane glycoproteins, gp100 contains a melanosome 1997;186:1213–1221. transport signal,18 which has been shown to traffic antigens to 9. Overwijk WW, Tsung A, Irvine KR, et al. gp100/pmel 17 is a the endocytic pathway for class II presentation.19 Because of murine tumor rejection antigen: induction of ‘‘self’’-reactive, tumoricidal T cells using high-affinity, altered peptide ligand. this, class II presentation of all endogenous antigens cannot J Exp Med. 1998;188:277–286. be presumed from the finding that transduced DCs are 10. Touloukian CE, Leitner WW, Topalian SL, et al. Identifi- capable of presenting a gp100-derived class II epitope. cation of a MHC class II-restricted human gp100 epitope However, endogenous antigens can be targeted to the class II using DR4-IE transgenic mice. J Immunol. 2000;164:3535– compartment by incorporation of sequences from the 3542. MHC class II–associated invariant chain20 or lysosomal- 11. Ito K, Bian HJ, Molina M, et al. 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Cancer Gene Therapy Antigen presentation by transduced dendritic cells JM Sloan et al 950 19. Wang S, Bartido S, Yang G, et al. A role for a melanosome ibility complex class II presentation of antigens. Proc Natl transport signal in accessing the MHC class II presentation Acad Sci USA. 1995;92:11671–11675. pathway and in eliciting CD4+ responses. J Immunol. 22. Zarling AL, Ficarro SB, White FM, Shabanowitz J, Hunt DF, 1999;163:5820–5826. Engelhard VH. Phosphorylated peptides are naturally pro- 20. Wang RF, Wang X, Atwood AC, Topalian SL, Rosenberg SA. cessed and presented by major histocompatibility complex Cloning genes encoding MHC class II-restricted antigens: class I molecules in vivo. J Exp Med. 2000;192:1755–1762. mutated CDC27 as a tumor antigen. Science. 1999;284:1351– 23. Haurum JS, Hoier IB, Arsequell G, et al. Presentation of 1354. cytosolic glycosylated peptides by human class I major histo- 21. Wu TC, Guarnieri FG, Staveley-O’Carroll KF, et al. compatibilitycomplex molecules in vivo. J Exp Med. 1999;190: Engineering an intracellular pathway for major histocompat- 145–150.

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