Leukemia (2001) 15, 1451–1457 2001 Nature Publishing Group All rights reserved 0887-6924/01 $15.00 www.nature.com/leu T cell activation following infection of primary follicle center lymphoma B cells with adenovirus encoding CD154 MJ Cantwell, WG Wierda, IS Lossos, R Levy and TJ Kipps Immunogenex, Inc., La Jolla, CA; Division of Oncology, Stanford University Medical School, Stanford, CA; and Division of Hematology/Oncology, UCSD School of Medicine, La Jolla, CA, USA Purified, high-titer adenovirus encoding murine CD154 (Ad- cell cytotoxicity towards nonmodified CLL B cells.13 More- CD154) or human CD154 (Ad-hCD154) was used to infect lymph over, a phase I clinical study using Ad-CD154 modified CLL node cells isolated from patients with follicle center lymphoma. 14 Infection of lymphoma B cells with Ad-CD154 at a multiplicity B cells has shown promising therapeutic results. Similarly, of infection (MOI) ratio of 100 or higher resulted in high-level other groups have shown that modification of various tumor transgene expression. Additionally, upon infection of lym- types to express CD154 can induce effective anti-tumor phoma B cells, only Ad-CD154 resulted in surface expression immune responses in animal tumor models.15–17 of CD154, despite similar, high-level expression of either These encouraging results stimulated us to examine the human or mouse CD154 by HeLa cells infected with Ad-hCD154 feasibility of Ad-CD154 immunotherapy for other B cell or Ad-CD154, respectively. Moreover, infection of lymphoma B cells with Ad-CD154, but not Ad-hCD154 or adenovirus enco- malignancies. B cell lymphoma appears to be an excellent ding Eschericheria coli beta-galactosidase (Ad-LacZ), induced candidate disease for this approach because, similar to CLL, the neoplastic B cells to express higher levels of immune co- lymphoma B cells express CD40 and can be induced to stimulatory molecules that are required for proficient presen- express multiple immune co-stimulatory molecules upon tation of antigen to T cells. Consistent with this, we found that CD40 ligation.18,19 Moreover, since lymphoma cells express Ad-CD154 infected lymphoma B cells could stimulate T cells to clonally restricted immunoglobulins and generally harbor proliferate or produce interferon-gamma in allogeneic or auto- logous mixed lymphocyte interactions. We conclude that cytogenetic abnormalities, these cells should express somati- lymphoma B cells can be infected with Ad-CD154 and that this cally generated antigens that potentially could be targeted by significantly enhances their recognition by allogeneic or auto- the host immune system.20 As such, we examined whether logous T cells. As such, Ad-CD154-transduced lymphoma B lymphoma B cells could be infected with recombinant aden- cells may have potential for the active immune therapy of ovirus vectors. Furthermore, we studied the phenotype and patients with follicle center lymphoma. Leukemia (2001) 15, immune function of such cells following infection with adeno- 1451–1457. Keywords: follicle center lymphoma; CD154; adenovirus virus encoding CD154. Materials and methods Introduction Patient samples CD40–CD154 (CD40-ligand) interactions are critical for immune recognition.1,2 CD154 is transiently expressed on + After obtaining informed consent, lymph node biopsies were CD4 T cells following T cell receptor (TCR) engagement by performed on patients with B cell follicle center lymphoma antigen-presenting cells through major histocompatibility and prepared into single cell suspensions, as described.21 The complex (MHC) class II molecules.3–6 This, in turn, can cause cells then were subjected to density-gradient centrifugation activation of CD40-expressing antigen presenting cells (APCs), over cushions of Ficoll–Paque PLUS (Amersham Pharmacia including B cells, dendritic cells, monocytes, and macro- Biotech, Uppsala, Sweden) to isolate mononuclear cells, as phages.7,8 Such CD40-activated cells can set off a cascade of per the manufacturer’s instructions. The cells were suspended immune-activating events that lead to a specific and effective in fetal calf serum containing 10% dimethylsulfoxide (Sigma, immune response against foreign antigens, such as viruses or St Louis, MO, USA) for storage in liquid nitrogen until use. tumors.9,10 The importance of CD40–CD154 interactions is Blood mononuclear cells were similarly prepared from either underscored by the finding that individuals who have normal donors or follicle center lymphoma patients as indi- inherited defects in the ligand for CD40 have a profound cated. Cell cultures were maintained in serum free AIM-V immune deficiency.11,12 Such patients have an immune media (Gibco-BRL, Grand Island, NY, USA). deficiency syndrome associated with impaired germinal center formation, defective isotype switching, and marked susceptibility to various bacterial and viral pathogens. Adenovirus vector constructs In light of the role that this receptor–ligand pair plays in immune regulation, the potential for using CD154 for immun- Replication-incompetent adenovirus vectors encoding murine otherapy is under active investigation. For example, chronic CD154 (Ad-CD154), human CD154 (Ad-hCD154), or ␤- lymphocytic leukemia (CLL) B cells modified to express galactosidase (Ad-LacZ) cDNA were made as described.13,22 CD154 using a replication defective adenovirus vector can Each cDNA was flanked by the cytomegalovirus (CMV) enhance CLL antigen presentation and induce autologous T promoter/enhancer on the 5Ј end and the bovine growth hor- mone polyadenylation signal sequence on the 3Ј end, allowing for high-level expression of the transgene mRNA in Correspondence: TJ Kipps, Division of Hematology/Oncology, Ј Department of Medicine, University of Californai-San Diego, 9500 eukaryotic cells without 3 non-translated regulatory 10 Gilman Dr., La Jolla, CA 92093-0663, USA; Fax: 858 534 5620 sequences. Purified, high-titer (eg 0.8–2 × 10 p.f.u./ml), Received 27 March 2001; accepted 13 May 2001 recombinant adenovirus was produced by Molecular Medi- T cell activation following infection of lymphoma B cells with Ad-CD154 MJ Cantwell et al 1452 cine, LLC (San Diego, CA, USA; www.molecularmed.com) by each well of a 96-well U-bottom plate. Subsequently, 105 pur- anion-exchange chromatography, as described.23–25 Adeno- ified blood T cells from allogeneic normal donors or 105 auto- virus purity and titers were determined by high performance logous blood T cells were added to each well. In each case, liquid chromatography (Figure 1) and by a plaque forming the CD3+ T cells were isolated to Ͼ93% purity using the T assay using 293 cells, as described.26 Cell Negative Isolation Kit from Dynal, Inc (Lake Success, NY, USA), by which CD19, CD14, CD16, CD56, and HLA-DR- positive cells were depleted. For autologous MLR reactions, Flow cytometry we added recombinant human interleukin-2 (IL-2; Biosource International, Cambridge, MA, USA) to each well to a final Cells were washed and suspended in staining buffer, com- concentration of 25ng/ml. For some samples, blocking-mAb posed of phosphate buffered saline (PBS, pH = 7.4) containing specific for MHC-class I (clone W6/32) was added at 3 ␮g/ml 3% fetal calf serum, 0.05% sodium azide, and 10 ␮g/ml pro- final concentration. Following 2 days’ incubation, aliquots of pidium iodide. Cells then were incubated with saturating the media were collected to measure IFN-␥ production by amounts of fluorochrome-conjugated monoclonal antibody enzyme-linked immunosorbent assay (ELISA) using two IFN- (mAb) for 30 min at 4°C. Stained cells were washed twice ␥-specific mAbs (Pharmingen) and the manufacturer’s proto- with staining buffer and analyzed using a FACSCaliber flow col. To assess cell proliferation, we added 3H-thymidine to a cytometer (Becton Dickinson, San Jose, CA, USA). Dead cells final concentration of 0.5 ␮Ci/well 5days after the initiation and debris were excluded from the analysis by light scatter of the MLR and then harvested the cells 12 to 16 h later to and by gating out cells that failed to exclude propidium iod- measure incorporated 3H-thymidine using a cell harvester ide. We determined the percentages of cells that expressed (Tomtec, Hamden, CT, USA) and a beta-scintillation counter the transgene product and the mean fluorescence intensity (Wallac, Turku, Finland). ratio (MFIR) of transgene-expressing cells to assess the relative expression levels of the transgene. The MFIR is calculated by dividing the mean fluorescence intensity (MFI) of cells that Results were stained with the fluorochrome-conjugated antigen-spe- cific mAb by the MFI of cells that were stained with a control Adenovirus infection of lymphoma cells fluorochrome-conjugated mAb of the same isotype, but of irrelevant specificity. All antibodies were obtained from We initially determined if replication-incompetent adenovirus Pharmingen, Inc. (La Jolla, CA, USA). These included fluor- encoding either murine CD154 (Ad-CD154) or human CD154 escein-conjugated mAb specific for CD3, CD27, CD70, and (Ad-hCD154) could infect lymphoma B cells and result in sur- CD80, and PE-conjugated mAbs specific for mouse CD154, face expression of CD154. Each vector was prepared to high- human CD154, CD86, CD95, and CD71. In addition, we used titer and purity, as assessed via anion-exchange chromato- CyChrome-conjugated mAb specific for HLA-DR and APC- graphy (Figure 1). Following infection with Ad-CD154 or Ad- conjugated mAb specific for CD19. hCD154 at increasing MOI ratios, HeLa cells expressed sur- face mouse or human CD154, respectively (Figure 2). More- over, the levels of surface CD154 expression on HeLa cells Mixed lymphocyte reactions were similar for either vector at the same relative MOI ratio (Figure 2). Likewise, infection of lymphoma cells with Ad- Two days after the initial infection with adenovirus, the CD154 also resulted in high expression levels of murine infected lymphoma stimulator cells were treated with 100 CD154 provided MOI ratios were у100 (Figure 2). However, ng/ml mitomycin C for 1 h at 37°C to block proliferation. infection of lymphoma cells with Ad-hCD154 did not result These cells then were washed three times to remove the mito- in significant expression of human CD154, even at MOI ratios mycin C.