Novel centrifugal method for simple and highly efficient adenovirus-mediated green fluorescence protein gene transduction into human monocyte-derived dendritic cells

Journal of Immunological Methods 253 Ž2001. 113–124 www.elsevier.nlrlocaterjim

Novel centrifugal method for simple and highly efficient adenovirus-mediated green fluorescence protein gene transduction into human monocyte-derived dendritic cells Naoki Nishimura, Yasuhiko Nishioka) , Tsutomu Shinohara, Hirohisa Ogawa, Sayaka Yamamoto, Kenji Tani, Saburo Sone Third Department of Internal Medicine, School of Medicine, UniÕersity of Tokushima, Kuramoto-cho 3, Tokushima 770-8503, Japan Received 17 October 2000; received in revised form 31 January 2001; accepted 21 February 2001

Abstract Dendritic cells ŽDC. are professional antigen-presenting cells in the immune system. Gene transduction of DC with tumor-associated antigen ŽTAA. or other genes that enhance the immune reaction has been considered theoretically useful for DC-based immunotherapy. However, gene transduction of DC generated from human peripheral blood monocytes has been difficult due to its low efficiency, even when adenoviral vector was used at high multiplicity of infection ŽMOI.. In the present study, we examined the effect of centrifugal force to enhance efficiency of adenovirus-mediated gene transduction into human monocyte-derived DC at various rotor speeds at various temperatures for various times. We judged the transduction efficiency using enhanced green fluorescence protein ŽEGFP.-expressing adenoviral vector, and the best condition for centrifugal transduction was determined as 2000 = g at 378C for 2 h at an MOI of 10 or greater. At an MOI of 50 without centrifugation, the gene transduction efficiency was about 66% and mean fluorescence intensity ŽMFI. of EGFP expression was about 150 Žat 378C for 2 h.. With centrifugal transduction Ž2000 = g at an MOI of 50 at 378C for 2 h., 86% or more DC were gene-modified, and especially, MFI of EGFP expression was highly enhanced ŽMFI: about 3100 or greater.. Centrifugally gene-transduced DC were not damaged and were thoroughly functional as measured by mixed lymphocyte reaction ŽMLR.. The centrifugal method was also applicable to human monocytes and K562 cells. The centrifugal transduction method with adenoviral vector might be helpful for the generation of gene-modified DC. q 2001 Elsevier Science B.V. All rights reserved. Keywords: Dendritic cells; Adenovirus vector; Centrifugation

AbbreÕiations: DC, dendritic cells; TAA, tumor-associated antigen; CTL, cytotoxic T cells; MOI, multiplicity of infection; PBMC, peripheral blood mononuclear cells; EGFP, enhanced green fluorescent protein; PFU, plaque forming units; mAbs, monoclonal antibodies; TdR, thymidine; MFI, mean fluorescence intensity; CAR, coxsackievirus and adenovirus receptor ) Corresponding author. Tel.: q81-88-633-7127; fax: q81-88633-2134. E-mail address: [email protected] ŽY. Nishioka..

1. Introduction Dendritic cells ŽDC. are professional antigen-presenting cells that play a major role in the immune system by stimulating naive T lymphocytes ŽInaba et al., 1990; Steinman, 1991; Liu and MacPherson, 1993; Hart, 1997.. DC have been used as a good vehicle for immunization against infectious ŽSteinman, 1991; Nair et al., 1993. and malignant diseases

0022-1759r01r$ - see front matter q 2001 Elsevier Science B.V. All rights reserved. PII: S 0 0 2 2 - 1 7 5 9 Ž 0 1 . 0 0 3 6 0 - X

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ŽMayordomo et al., 1995; Porgador and Gilboa, 1995; Porgador et al., 1996; Hsu et al., 1996.. In general, isolated or ex vivo-generated DC pulsed with antigen peptides or tumor lysate have been inoculated into cancer patients. Recently, much attention has been paid to gene modification of DC with tumor-associated antigen ŽTAA. which could provide a more extended duration of antigen presentation and induce antigenspecific cytotoxic T cells ŽCTL. more efficiently than peptide pulsing ŽReeves et al., 1996; Specht et al., 1997; Wan et al., 1997; Butterfield et al., 1998; Perez-Dıez ´ ´ et al., 1998; Kirk and Mule, ´ 2000.. Furthermore, transduction of other genes such as cytokines or chemokines into DC could also enhance the immune reaction in DC-based immunotherapy ŽNishioka et al., 1999; Kirk and Mule, ´ 2000.. In previous studies, retrovirus- or adenovirus-mediated gene transfer into DC has been examined to enhance the efficiency of gene transduction compared with non-viral gene delivery. However, the transduction efficiency into human DC, which are differentiated and postmitotic cells, was usually low using retroviral vector systems because transfer of genes by retroviral vectors needs proliferation of the target cells to attain integration and stable transgene expression. Even the adenoviral vector, which is capable of targeting non-proliferating cells, requires high doses of viruses, such as multiplicity of infection ŽMOI. of 100 or 1000, for sufficient gene transduction into human monocyte-derived DC ŽArthur et al., 1997; Butterfield et al., 1998; Perez-Dıez ´ ´ et al., 1998.. In the present study, we report that the efficiency of the adenovirus-mediated marker-gene transduction into human monocyte-derived DC was remarkably enhanced by centrifugal manipulation, which is very simple and reproducible. 2. Materials and methods 2.1. Reagents and cell lines Culturing of DC was performed in RPMI 1640 ŽNissui Chemical, Tokyo, Japan. supplemented with 5% heat-inactivated human AB serum Žobtained from healthy donors., 2 mM L-glutamine ŽGIBCO, Grand Island, NY., 1 mM MEM sodium pyruvate solution ŽGIBCO, Grand Island, NY., 0.1 mM MEM non-es-

sential amino acids solution ŽGIBCO, Grand Island, NY. and 50 mgrml gentamycin ŽSchering-Plough, Osaka, Japan.. Culturing of monocytes, lymphocytes, tumor cells and MLR assays were performed in RPMI 1640 ŽNissui Chemical, Tokyo, Japan. supplemented with 10% heat-inactivated FBS ŽGIBCO, Grand Island, NY., and 50 mgrml gentamycin ŽSchering-Plough, Osaka, Japan.. Human recombinant GM-CSF Žspecific activity: 1 = 10 8 Urmg. was from Kirin Brewery ŽTokyo, Japan.. Human recombinant IL-4 Žspecific activity: 1 = 10 6 Urmg. was from Ono Pharmaceuticals ŽOsaka, Japan.. Human serum albumin Ž20%. was purchased from Takeda Pharmaceuticals ŽOsaka, Japan.. None of these materials contained endotoxins, as judged by the Limulus amebocyte assay Žsensitivity limit, 0.1 ngrml. ŽSeikagaku Kogyo, Tokyo, Japan.. 2.2. Isolation of monocytes and lymphocytes Leukocyte concentrates from healthy donors were separated into peripheral blood mononuclear cells ŽPBMC. by density gradient centrifugation in lymphocyte separation medium. Then, PBMC were separated into lymphocytes and monocytes by counterflow centrifugal elutriation in a Beckman JE-5.0 rotor ŽBeckman Instruments, Fullerton, CA. by the method described previously ŽMaeda et al., 1991.. The lymphocyte-rich and monocyte-rich fractions were collected at flow rates of 12–16 and 17–20 mlrmin, respectively, at 2000 rpm. The purity of the monocyte fraction, determined by morphologic examination and non-specific esterase staining, was greater than 95%, and that of the lymphocyte fraction was greater than 99%. 2.3. Generation of DC For the generation of monocyte-derived DC ŽNishimura et al., 2000., monocytes were cultured in six-well tissue culture plates Ž2 = 10 6 cellsrwell. ŽBecton Dickinson Labware, Franklin Lakes, NJ. in medium containing cytokines wGM-CSF Ž500 Urml. q IL-4 Ž250 Urml.x. On day 3, the cultures were fed with fresh medium containing cytokines and incubated for another 3 days. After 6 days of culture, only non-adherent cells were harvested, washed with PBS and used for the following experiments as DC.

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Fig. 1. Transduction efficiency at various centrifugal forces. DC were transduced with AxCAegfp at an MOI of 50 at 378C for 2 h. Centrifugal forces were set at 300= g, 1000= g, and 2000= g. For the non-centrifugally transduced control, DC were incubated in a 5% CO 2 incubator. Expression of EGFP was evaluated by flow cytometry 24 h after gene transduction. MFI: mean fluorescence intensity. Scale bars and the numbers indicated under the scale bars: EGFP-expressing cells and the percentage of gated cells that were EGFP-expressing, respectively.

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Fig. 2. Transduction efficiency at various temperatures. DC were transduced with AxCAegfp at an MOI of 50 at 2000= g for 2 h. Transduction temperatures of 228C, 308C, and 378C were tested. Expression of EGFP was evaluated by flow cytometry 24 h after gene transduction. MFI: mean fluorescence intensity. Scale bars and the numbers indicated under the scale bars: EGFP-expressing cells and the percentage of gated cells that were EGFP-expressing, respectively.

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All of the following functional studies were performed after removal of the cytokines. 2.4. AdenoÕiral Õector construction and preparation We used the recombinant adenovirus AxCAegfp, which contained an enhanced green fluorescent protein ŽEGFP. gene. For adenovirus preparation, an Adenovirus Expression Vector Kit ŽTaKaRa Shuzo, Kyoto, Japan. was used, in which adenoviral cosmid, pAxCAwt, was included ŽKanegae et al., 1995.. The cosmid pAxCAwt consisted of E1- and E3-deficient adenovirus type 5 ŽAd5. sequences, and the CAG promoter ŽNiwa et al., 1991. and rabbit b-globin poly A were inserted at the former E1 site in reverse orientation with respect to the Ad5 sequences. EGFP cDNA was obtained from pEGFP-N1 ŽClontech, Palo Alto, CA. ŽCormack et al., 1996. and subcloned into pAxCAwt. The resulting cosmid was named pAxCAegfp. Transfection of 293 cells with pAxCAegfp was performed as described previously ŽMiyake et

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al., 1996. and produced replication-incompetent, E1and E3-deficient adenoviral vector expressing EGFP. Recombinant virus ŽAxCAegfp. was prepared by expansion of a single clone generated in 293 cells, which was purified by limiting dilution and was fluorescent. There was no detectable replicationcompetent adenovirus or E1q virus in the preparation. 2.5. AdenoÕirus-mediated gene transfer into DC The adenovirus was suspended in PBS supplemented with 1% human serum albumin ŽHSArPBS. and adjusted to 1 = 10 8 plaque forming units ŽPFU.rml or 2 = 10 7 PFUrml. DC or other targeted cells were harvested, washed once in PBS and resuspended at 2 = 10 6 cellsrml in HSArPBS. Then, 500 ml of cells Ž1 = 10 6 cells. were mixed with 500 ml of the adenovirus ŽMOI of 50 or 10. and 1 ml of the mixture was poured into a polypropylene tube ŽFALCON 2059; Becton Dickinson Labware, Lin-

Fig. 3. Transduction efficiency at various transduction times. DC were transduced with AxCAegfp at an MOI of 10 at 2000 = g at 378C. Transduction times of 15, 30 min, 1, 2, and 3 h were tested. Control means non-transduced control. Expression of EGFP was evaluated by flow cytometry 24 h after gene transduction. MFI: mean fluorescence intensity. Viable cells: the percentage of viable cells evaluated by trypan blue staining 24 h after gene transduction. Scale bars and the numbers indicated under the scale bars: EGFP-expressing cells and the percentage of gated cells that were EGFP-expressing, respectively.

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coln Park, NJ.. For the centrifugal transduction, the tubes were centrifuged at various centrifugal forces at various temperatures for various times in an LIX130 temperature-controlled centrifuge ŽTomy, Tokyo, Japan.. For the non-centrifugal control, the tubes were incubated at 378C in a 5% CO 2 incubator or at room temperature for 2 h. For the non-transduced control, 500 ml of cells were mixed with 500 ml of virus-free HSArPBS and 1 ml of the mixture was poured into a FALCON 2059 tube. Then the tube was incubated at room temperature for 2 h. After incubation for gene transfer, the cell and virus mixture was washed twice in PBS. DC were resuspended at 5 = 10 5 cellsrml in medium containing cytokines wGM-CSF Ž500 Urml. q IL-4 Ž250 Urml.x and cultured in six-well tissue culture plates ŽBecton Dickinson Labware, Franklin Lakes, NJ.. Monocytes, lymphocytes and tumor cells were resuspended in medium without cytokines. After 24 h of

culture, cells were harvested and used for the following experiments. 2.6. Flow cytometric measurement of EGFP expression and surface antigen expression The harvested cells were washed once and EGFP expression was quantified by flow cytometry using a FACSCalibur flow cytometer with CellQuest software ŽBecton Dickinson, San Jose, CA.. EGFP absorbs light energy at 488 nm and emits light at 507 nm, which was detected by the FL1 detector of the FACSCalibur. For measurement of surface antigen expression, the harvested DC were washed once and incubated for 30 min at 48C in PBS containing the primary mAb Ž2 mgrml., as indicated below. Cells were then indirectly labeled with the RPE-Cy5-conjugated FŽabX . 2 fragment of rabbit anti-mouse immunoglobulins ŽDAKO, Glostrup, Denmark. Ž25 mgrml,

Fig. 4. Cell viabilities and EGFP expression after 6 days of culture. DC were transduced with AxCAegfp at an MOI of 50 at 378C for 2 h with or without centrifugation Ž2000 = g .. Control means non-transduced control. Expression of EGFP was evaluated by flow cytometry 24 h or 6 days after gene transduction. MFI: mean fluorescence intensity. Viable cells: the percentage of viable cells evaluated by trypan blue staining. Scale bars and the numbers indicated under the scale bars: EGFP-expressing cells and the percentage of gated cells that were EGFP-expressing, respectively.

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48C for 30 min. and analyzed by flow cytometry. Light energy absorbed at 488 nm by RPE was transferred to Cy5, which emits light at 670 nm, which was detected by the FL3 detector of the FACSCalibur. The fluorescence of EGFP Žmaximum emission: 507 nm. and RPE-Cy5 Žmaximum emission: 670 nm. interfered very little with each other, so that the coexpression of EGFP and the surface antigen could be distinguished clearly. The following monoclonal antibodies ŽmAbs. were used for the primary mAbs: anti-CD1a, -CD83 and -HLA-A, B, C were obtained from Immunotech ŽMarseilles, France.. Anti-CD14 was obtained from Sanbio ŽUden, Netherlands.. Anti-CD54, -CD80, -CD86 and -HLA-DR were purchased from Pharmingen ŽSan Diego, CA.. All figures and table show representative findings from several independent experiments. 2.7. Mixed lymphocyte reaction (MLR) Four hundred thousand allogeneic lymphocytes were plated per well in 96-well tissue culture plates ŽCorning Glass Works, Corning, NY. and cultured with different numbers of DC in triplicate. After 96 h

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of incubation, proliferation of lymphocytes was determined by measuring 3 H-thymidine ŽTdR. incorporation. Briefly, 3 H-TdR Ž1 mCirwell. was added to the cultures, and after an additional 18 h of incubation, 3 H-TdR uptake was measured using a liquid scintillation counter. All values are the representative findings from several independent experiments. 3. Results 3.1. Efficiency of the centrifugal adenoÕirus transduction into human monocyte-deriÕed DC under Õarious conditions To optimize the conditions of the centrifugal adenovirus transduction, we examined the centrifugal transduction at various rotor speeds at various temperatures for various times. At first, we examined the transduction efficiency at various rotor speeds. We fixed the transduction temperature at 378C, transduction time at 2 h and MOI at 50. As shown in Fig. 1, with increasing centrifugal force, the transduction efficiency in-

Fig. 5. Surface marker expression of AxCAegfp-transduced DC. Surface marker expression was analyzed by flow cytometry 24 h after adenovirus transduction. Dotted line: non-transduced control DC. Bold line: DC transduced with AxCAegfp at an MOI of 10 at 378C for 2 h in combination with centrifugation Ž2000 = g .. MFI: mean fluorescence intensity. All data are not gated on EGFP positive cells.

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creased and, especially, mean fluorescence intensity ŽMFI. increased remarkably. The most effective centrifugal force tested was 2000 = g. The centrifuge used could only reach a centrifugal force of 2060 = g, so we could not examine higher centrifugal forces than 2000 = g. Next, we examined the transduction efficiency at various temperatures. We used a centrifugal force of 2000 = g, a transduction time of 2 h and MOI of 50. As shown in Fig. 2, about 90% of DC expressed EGFP irrespective of the transduction temperature. However, with an increase in the transduction temperature, MFI increased remarkably, and 378C was the most effective temperature. Finally, we examined the transduction efficiency at various transduction times. We used a centrifugal force of 2000 = g, a transduction temperature of 378C, and MOI of 10. As shown in Fig. 3, with increasing transduction time, the percentage of EGFP expressing DC and their MFI increased. We also evaluated the viabilities of centrifugally gene-transduced DC by trypan blue dye-exclusion 24 h after gene transduction. Although the transduction efficiency was highest with a 3-h transduction, fewer viable cells were harvested than after a 2-h centrifugation or less ŽFig. 3.. The most effective transduction time was therefore considered to be 2-h. Thus, it was suggested that the best condition of the centrifugal transduction was at 2000 = g at 378C for 2 h. With these conditions, approximately 77% of DC were transduced only at an MOI of 10 ŽFig. 3. and greater than 86% DC were transduced at an MOI of 50 ŽFigs. 1 and 2. by the centrifugal method. We also estimated the viabilities of centrifugally transduced DC Ž2000 = g at 378C for 2 h at an MOI of 50. and non-transduced control DC by trypan blue dye-exclusion until day 6 after gene transduction. There were no differences in the percentage of viable DC between the transduced and control DC ŽFig. 4.. EGFP expression of centrifugally transduced DC was still as high on day 6 after gene transduction as on day 1 ŽFig. 4.. 3.2. Phenotype of the DC centrifugally transduced with adenoÕirus To determine whether adenovirus-transduced DC had a mature DC phenotype, we analyzed the expression of various surface antigens by flow cytometry

of DC centrifugally transduced with adenovirus Žat an MOI of 10 at 2000 = g at 378C for 2 h. and non-transduced control DC ŽFig. 5.. There were no remarkable changes in the expression of CD1a, CD14, CD80, CD83 and CD86 between control DC and adenovirus-transduced DC. In contrast, adenovirus-transduced DC expressed slightly higher levels of MHC molecules ŽHLA-class I and HLA-DR. than control DC. Overall, these findings indicate that adenovirus-mediated gene transduction of DC had little effect on the ability of DC to express appropriate cell surface markers. 3.3. Antigen-presenting ability of the DC centrifugally transfected with adenoÕirus To test whether adenovirus gene-transduced DC retained their ability to stimulate T-cell proliferation,

Fig. 6. Allo-stimulatory capacity of AxCAegfp-transduced DC. DC were transduced with AxCAegfp at an MOI of 10 at 378C for 2 h with or without centrifugation Ž2000= g .. Allogeneic lymphocytes were stimulated by DC at different LymphocytesrDC ratios Žallo-MLR.. For MLR, three kinds of DC, which are indicated as Non-transduced control, Non-centrifugal control and Centrifugation, were used as stimulators. Four hundred thousand allogeneic lymphocytes were plated per well in 96-well tissue culture plates and cultured with different quantities of DC in triplicate. The proliferation of lymphocytes was measured by 3 H-thymidine ŽTdR. incorporation on day 4 of culturing. The degree of proliferation is indicated as cpm. Black bars: ten thousand DC per well. Hatched bars: two thousand DC per well. White bars: control value of lymphocytes only. †: Mean"SD of triplicate cultures.

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Table 1 EGFP expression of various non-adherent cells transduced with AxCAEGFP Human monocytes

Control c 378C d Centrifugatione

Human lymphocytes K562

H69

N291

Transduced cells a Ž%.

MFI b

Transduced cells Ž%.

MFI

Transduced cells Ž%.

MFI

Transduced cells Ž%.

MFI

Transduced cells Ž%.

MFI

0.09 0.79 26.80

7.60 9.55 292.36

0.90 1.12 1.45

6.33 6.19 7.20

0.88 43.64 60.92

10.55 105.45 1633.37

0.00 2.19 1.57

8.39 19.73 47.30

0.01 6.04 3.58

10.05 80.21 96.21

a

EGFP expression was measured by flow cytometry. The percentage of FL1 ŽEGFP. positive cells was expressed as transduced cells. MFI: Mean fluorescence intensity ŽMFI. of FL1 histogram. c Control: Non-transduced control cells. d 378C: AxCAEGFP was transduced at a MOI of 50 at 378C for 2 h in a 5% CO 2 incubator. e Centrifugation: AxCAEGFP was transduced at a MOI of 50 at 378C for 2 h in combination with centrifugation Ž2000 = g .. b

we performed allogeneic MLR. As shown in Fig. 6, EGFP-transduced DC showed similar or a slightly higher allo-stimulatory capacity compared with nontransduced DC and DC transduced without centrifugation. These results at least showed that adenoviral transduction did not reduce allogeneic stimulatory capacity. 3.4. AdenoÕirus-mediated gene transduction of human monocytes, lymphocytes and Õarious cell lines We also examined the centrifugal transduction of adenoviral vector into human monocytes, lymphocytes and some cell lines. In human monocytes, the centrifugal method enhanced the gene transduction efficiency ŽTable 1.. However, adenovirus transduction into human lymphocytes was very inefficient, and centrifugal enhancement was not observed ŽTable 1..

4. Discussion In the present study, we showed that the efficacy of adenovirus-mediated EGFP gene transduction into DC was markedly enhanced by centrifugation during transduction, and the best conditions for centrifugal transduction were determined. We used EGFP-expressing adenovirus vector as a marker for gene transduction. Expression of EGFP can easily be confirmed using fluorescent microscopy or flow cytometry. Flow cytometric analysis can estimate the accurate ratio of transduction efficiency. Gene trans-

duction of EGFP might be beneficial for evaluation of gene-transduction efficiency compared with transduction of other marker genes such as b-gal gene and so on. One of the limitations of the clinical application of DC-based cancer immunotherapy is the difficulty of preparing a sufficient number of human DC. Monocyte-derived DC might have many benefits in clinical trials because they are easier to generate in vitro or ex vivo than other lineages of DC. Furthermore, genetic modification of monocyte-derived DC is expected to enhance the antigen-presenting function of the DC. Indeed, there are many studies describing gene delivery to DC. However, non-viral gene delivery, including that achieved with CaPO4 , cationic liposomes, and electroporation, could not achieve adequate transduction efficiency for clinical application ŽArthur et al., 1997; Dietz and VukPavlovic, ´ 1998.. The retrovirus system also could not achieve a high transduction efficiency for monocyte-derived DC since DC are differentiated, postmitotic cells. Recently, DC transduced with the TAA gene using adenovirus vector was reported to have effectively induced TAA-specific CTL ŽWan et al., 1997; Butterfield et al., 1998; Perez-Dıez ´ ´ et al., 1998.. Gene modification of DC with the TAA gene has the potential to present various known and unknown TAA epitopes on MHC molecules of DC. Moreover, endogenous processing and presentation of TAA peptides may be more efficient for cell surface presentation than exogenous loading of synthetic TAA peptides ŽHahn et al., 1996.. However, sufficient gene transduction of monocyte-derived DC required high doses of adenoviruses, such as an MOI

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of 100 or 1000 ŽArthur et al., 1997; Butterfield et al., 1998; Perez-Dıez ´ ´ et al., 1998., which caused cell damage. Thus, the centrifugal transduction method might be beneficial. Some previous studies showed the methods of highly efficient gene transduction into DC. Dietz and Vuk-Pavlovic´ showed adenoviral transduction into DC were highly enhanced in combination with cationic liposomes. However, cationic liposomes sometimes cause cell damage. Ponsaerts et al. Ž2000. reported that mRNA could be efficiently transduced into DC using electroporation. However, mRNA is not easy to treat because their stability is low. Therefore, the centrifugal method would be more advantageous in clinical use. Centrifugation has been used for many years to enhance infection of cultured cells with various types of viruses, such as cytomegalovirus ŽHodgkin et al., 1988. and retrovirus ŽHo et al., 1993; Kotani et al., 1994; Bahnson et al., 1995.. The present findings showed that the transduction efficiency increased with increasing centrifugal force. However, the reason why centrifugal force can enhance the transduction efficiency is not understood. We speculate that centrifugal force might enhance the chance of virus particles to attach with their cellular receptor, coxsackievirus and adenovirus receptor ŽCAR. and integrins a V b 3 and a V b 5 ŽWickham et al., 1993; Mathias et al., 1994; Bergelson et al., 1997.. The detailed mechanism of the centrifugal enhancement should be examined in future studies. The temperature of virus infection is one of the key parameters determining the infection efficiency. For example, retroviral vectors are transduced most efficiently at 328C, probably because retroviruses are more stable at 328C than 378C ŽKotani et al., 1994.. Adenovirus-mediated gene transduction is considered to be more efficient at 378C than at lower temperatures ŽPellegrini et al., 1998.. This was confirmed and extended by our present findings showing that 378C was the most effective temperature when combined with centrifugation. High temperature may cause high metabolism of DC and increased internalization of adenoviruses. Increasing the time of adenovirus-mediated gene transduction in combination with centrifugation resulted in higher efficiency of gene transduction. However, centrifugal transduction for 3 h was found to be damaging at 2000 = g at

378C. The centrifuge used could only reach a centrifugal force of 2060 = g at 378C, so we could not examine higher centrifugal forces than 2000 = g. However, we thought that the best condition of the centrifugal transduction was at 2000 = g at 378C for 2 h. As mentioned, 3-h centrifugal transduction caused cell damage. Like this, this condition Žat 2000 = g at 378C for 2 h. was considered one of the limits of centrifugal force against DC. Moreover, we could achieve almost 90% transduction efficiency at 2000 = g at 378C for 2 h at an MOI of 50. We considered the transduction efficiency was enough for clinical use. Thus, it was suggested that the optimal conditions for the centrifugal transduction of adenoviral vector into DC was: at 2000 = g at 378C for 2 h at an MOI of 10 or greater. The findings of the phenotypic analysis of the DC transduced by the centrifugal method showed that they expressed slightly higher levels of MHC than that of non-transduced control DC. It was suggested that the slightly enhanced expression of MHC by adenovirus-transduced DC might make them more competent antigen presenting cells. These might be among the advantages of the adenoviral gene transduction of DC. DC transduced with the centrifugal method showed similar or a slightly higher allostimulatory capacity compared with control DC. Higher expression of MHC molecules of centrifugally transduced DC might promote the T-cell proliferation, or endogenous expression of EGFP might result in promoting the endogenous processing and presentation of EGFP antigen peptides and in enhancing MLR. We showed that the centrifugal method also enhanced the efficiency of adenovirus-mediated gene transduction into human monocytes and K562 cells. However, adenovirus transduction of human lymphocytes was very inefficient, and centrifugal enhancement was not observed. Adenovirus transduction of H69 cells and N291 cells also showed low efficiency, and centrifugal enhancement was minimal. Human lymphocytes may have few adenovirus receptors, as reported by Leon et al. Ž1998.. Monocyte lineage cells, such as monocytes and monocyte-derived DC, and K562 cells might have more adenovirus receptors. Finally, the centrifugal method might be beneficial for generation of gene-modified DC as well as

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gene transduction into other types of cells that have adenovirus receptors. The centrifugal adenovirus transduction is very simple, highly efficient, and not cytopathic, and, therefore, many applications, including use in clinical trials, are anticipated. Further investigations of the transduction of TAA or cytokine gene into DC by the centrifugal method are ongoing in our laboratory. Acknowledgements We thank Ms. Fumie Kaneko for assistance in the preparation of monocytes and lymphocytes. This work was supported by Grants-in-aid for Cancer Research from the Ministry of Education, Science, Culture and Sports of Japan. References Arthur, J.F., Butterfield, L.H., Roth, M.D., Bui, L.A., Kiertscher, S.M., Lau, R., Dubinett, S., Glaspy, J., McBride, W.H., Economou, J.S., 1997. A comparison of gene transfer methods in human dendritic cells. Cancer Gene Ther. 4, 17. Bahnson, A.B., Dunigan, J.T., Baysal, B.E., Mohney, T., Atchison, R.W., Nimgaonkar, M.T., Ball, E.D., Barranger, J.A., 1995. Centrifugal enhancement of retroviral mediated gene transfer. J. Virol. Methods 54, 131. Bergelson, J.M., Cunningham, J.A., Droguett, G., Kurt-Jones, E.A., Krithivas, A., Hong, J.S., Horwitz, M.S., Crowell, R.L., Finberg, R.W., 1997. Isolation of a common receptor for coxsackie B viruses and adenoviruses 2 and 5. Science 275, 1320. Butterfield, L.H., Jilani, S.M., Chakraborty, N.G., Bui, L.A., Ribas, A., Dissette, V.B., Lau, R., Gamradt, S.C., Glaspy, J.A., McBride, W.H., Mukherji, B., Economou, J.S., 1998. Generation of melanoma-specific cytotoxic T lymphocytes by dendritic cells transduced with a MART-1 adenovirus. J. Immunol. 161, 5607. Cormack, B.P., Valdivia, R.H., Falkow, S., 1996. FACS-optimized mutants of the green fluorescent protein ŽGFP.. Gene 173, 33. Dietz, A.B., Vuk-Pavlovic, ´ S., 1998. High efficiency adenovirusmediated gene transfer to human dendritic cells. Blood 91, 392. Hahn, Y.S., Hahn, C.S., Braciale, T.J., 1996. Endogenous presentation of a nascent antigenic epitope to CD8q CTL is more efficient than exogenous presentation. Immunol. Cell. Biol. 74, 394. Hart, D.N.J., 1997. Dendritic cells: unique leukocyte populations which control the primary immune response. Blood 90, 3245. Ho, W.Z., Cherukuri, R., Ge, S.D., Cutilli, J.R., Song, L., Whitko, S., Douglas, S.D., 1993. Centrifugal enhancement of human

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