Spontaneous transgenesis of human B lymphocytes

Gene Therapy (2004) 11, 42–51 & 2004 Nature Publishing Group All rights reserved 0969-7128/04 $25.00 www.nature.com/gt

RESEARCH ARTICLE

Spontaneous transgenesis of human B lymphocytes G Filaci1, M Gerloni1,2, M Rizzi1, P Castiglioni1, H-D Chang1, MC Wheeler1, R Fiocca3 and M Zanetti1 1

The Department of Medicine and Cancer Center, University of California, San Diego, La Jolla, CA, USA; 2Cosmo Bioscience, La Jolla, CA, USA; and 3The Department of Surgical and Morphological Sciences, Division of Human Pathology, University of Genova, Italy

DNA can cross the cell membrane by natural means, but the functional relevance of this phenomenon has not been fully elucidated. Here, we analyzed spontaneous transgenesis of human B cells using plasmid DNA coding for a functional immunoglobulin (Ig) heavy chain gene under the control of a B-cell-specific promoter. Using polymerase chain reaction (PCR), reverse transcriptase-PCR, and flow cytometry in combination, spontaneous transgenesis was documented in Burkitt’s lymphoma cell lines, Epstein–Barr virus-transformed cell lines, and peripheral blood B lymphocytes of the mature naı¨ve phenotype (IgM þ /IgD þ /CD27). By immunoelectron microscopy, the internalized DNA was seen in the lysosomes/late endosomes and in the cytosol proximal to the

nucleus. Importantly, spontaneously transgenic B cells processed and presented to major histocompatibility complex (MHC)-restricted T lymphocytes a peptide expressed in the transgenic product. This is the first demonstration that primary B lymphocytes possess a program for the spontaneous internalization of DNA, which in turn imparts the cell with new immunological functions. As spontaneous transgenesis is obtained using a nonviral vector, does not require prior cell activation, and is not associated with chromosomal integration, the findings reported here open new possibilities for genetic manipulations of mature naı¨ve B lymphocytes for therapy and vaccination. Gene Therapy (2004) 11, 42–51. doi:10.1038/sj.gt.3302132

Keywords: spontaneous transgenesis; plasmid DNA; B lymphocytes; antigen presentation

Introduction Gene manipulations by transfer or insertion into living cells play a relevant role in today’s medical sciences and apply to gene therapy,1 gene vaccination,2 and therapeutic cloning.3 In these instances, gene transfer across the cell membrane involves either transfer of whole chromosomal nuclear content into a host cell,4 cell transfection with artificial chromosomes,5,6 receptor-mediated endocytosis of viral vectors,7,8 or transfer of plasmid DNA coding for a single functional gene or an assortment of gene segments.9 In nature, cell transfer of genetic material is generally facilitated by specific receptors or structures on microorganisms,10–12 but it can also occur spontaneously without carrier enhancement. In fact, early reports showed the capacity of eucaryotic cells to incorporate exogenous DNA13–16 and acquire new phenotypic characteristics.17 As these studies were performed at a time when sensitive methods of DNA analysis and amplification were not yet available, the phenomenon of spontaneous transgenesis was not analyzed further. A resurgence of interest in spontaneous transgenesis of mammalian cells18 came with the Correspondence: M Zanetti, University of California, San Diego, 9500 Gilman Dr, La Jolla, CA 92093-0837, USA The first two authors contributed equally to this work Current address of GF: Division of Internal Medicine and Clinical Immunology, Department of InternalMedicine and Center of Excellence for Biomedical Research, University of Genova, Italy Current address of H-DC: Deutsches Rheumaforschungszentrum, 10115 Berlin, Germany Received 16 April 2003; accepted 10 July 2003

demonstration that the injection of plasmid DNA intramuscularly or intradermally yields systemic immune responses,19,20 yet the mechanisms of transgenesis and which cells are susceptible to DNA uptake in vivo remain largely unknown.21 The present study began with the intention to gain insights into the possible downstream functional effects of spontaneous transgenesis in lymphoid cells. As a model system we used bacterial DNA in the form of a plasmid containing a functional immunoglobulin (Ig) heavy (H) chain gene (the transgene) regulated by a B-cell-specific promoter and human B cells as target. Systematically, we documented internalization of plasmid DNA, gene transcription and translation, and intracellular processing of the transgene product. Owing to the model system chosen, key immunological functions such as processing and presentation of peptides of the transgenic product to T lymphocytes could be conveniently used as a readout of the downstream events of spontaneous transgenesis.

Results We analyzed transgenesis in the following types of human B cells: Burkitt’s lymphoma cell lines, Epstein– Barr virus (EBV)-transformed lymphoblastoid cells, and peripheral blood lymphocytes (PBL). The cells were invariably transfected by incubation with plasmid DNA in phosphate-buffered saline (PBS) solution without Ca2 þ and Mg2 þ for 60 min. After incubation, the cells were washed and cultured overnight in medium at 371C. With the exception of experiments shown in Figure 6,

Transgenesis of human B lymphocytes G Filaci et al

103

104

a

105

Cell number

DNA (µg 5x)

b

12 hrs

BJAB

24 hrs

EBV

48 hrs

PBL

(+) control

100 10-1 10-2 10-3 10-4 (-) control

~200 bp

c

(-) control

Intracellular compartmentalization of plasmid DNA in spontaneously transfected PBL In light of the result shown in Figure 1c we used immunoelectron microscopy to monitor entry and intracellular compartmentalization of plasmid DNA in PBL from normal donors. First, we assessed the sensitivity and specificity of detection using a monoclonal antibody coupled with gold particles. The number of gold particles was counted in the cytoplasm of PBL incubated with the plasmid and compared with what was present in control PBL. Examining 24 consecutive fields averaging a total cytoplasmic area of 16 319 mm2 (transgenic PBL) and 16 126 mm2 (control PBL), we counted 376 gold particles in transgenic PBL compared with 51 gold particles in control PBL (Po0.001) (Table 1). Comparable numbers and localization of gold particles were observed in all PBL preparations analyzed. No gold particles were observed outside the cells. At 1 h after spontaneous transgenesis, electron micrographs showed the presence of DNA molecules inside the cell membrane in three cellular districts: endosomal vesicles (Figure 2a and b), lysosomes (Figure 2c), and cytosol (Figure 2d). Using immunogold beads of different sizes, internalized DNA was localized in endosomal vesicles also containing human lymphocyte antigen (HLA) class II molecules (Figure 2b – double arrow). Molecules of DNA were also detected in the proximity of the outer side of the nuclear membrane (Figure 2d and e), suggesting imminent translocation.25 Previous studies showed that uptake of exogenous DNA by cells in culture is a function of time and concentration of input DNA,14 and that only a small fraction persists in the cells 24 h after internalization.13–15 This indicates that the major fraction of internalized DNA is degraded intracellularly. Not surprisingly, we observed plasmid DNA in vesicle structures with morphological characteristics of lysosomes (Figure 2c), likely implying DNA degradation.

(+) control

Spontaneous transfection in human B cells Transfection was first analyzed using a Burkitt’s lymphoma cell line (Raji) at different time intervals after contact with plasmid DNA. The Ig transgene was amplified from cellular DNA by a polymerase chain reaction (PCR) using a set of primers (pCL/pCD) designed to anneal two sites within the VDJ region (E566 bp) and followed by a round of nested PCR using a set of primers (pNED/pNEL) amplifying a fragment of 200 bp as described previously.24 A product corresponding to the rearranged V region was consistently amplified 12–48 h after transfection (Figure 1a), suggesting that uptake of exogenous DNA is very rapid. In follow-up studies, no PCR product could be detected by the third week after transfection (not shown). A PCR product was amplified at each time point through 102 cells/well (Figure 1a). Spontaneous transfection was possible using as little as 5  104 mg of plasmid/105 cells (Figure 1b). This phenomenon was not unique to Raji cells since using BJAB cells, another Burkitt lymphoma cell line, a (+) control

PCR product was similarly amplified 24 h after transfection (Figure 1c – upper panel). To rule out the possibility that spontaneous transfection requires a neoplastic phenotype (ie, cells with a high proliferation rate and genetic abnormalities that could favor integration), we used two non-neoplastic B-cell types: EBV-transformed lymphoblastoid cells and lymphocytes in peripheral blood of normal donors. PCR analysis of cellular DNA extracted 24 h after the incubation of cells with plasmid DNA revealed a transgene-specific amplification in both the cell types (Figure 1c, mid and lower panels). This suggests that susceptibility to spontaneous transfection does not require a neoplastic phenotype.

102 (-) control

all experiments were performed with plasmid DNA (15.3 kb) coding for an Ig H chain gene composed of the rearranged murine VH62 gene,22 a member of the VH7183 gene family, and the human g1 constant region. Plasmid g1NANP carries an insertion in the third complementarity-determining region (CDR3) of the variable domain coding for three repeats of the tetrapeptide NANP.22 Plasmid g1MART (used in experiments shown in Figure 6) codes for the amino-acid sequence AAGIGILTV (position 27–35) from the MART-1 melanoma-associated antigen23 in lieu of the three NANP repeats. Both plasmids are under the control of a B-cell-specific promoter.

Figure 1 Detection of the transgene after spontaneous transgenesis. Nested PCR of the g1NANP transgene in cellular DNA extracted from (a and b) Raji cells and (c) BJAB cells, EBV-transformed lymphoblastoid cells, and human PBL. (a) Nested PCR amplification was performed 12 (upper row), 24 (middle row), and 48 (lower row) h after transgenesis on DNA extracted from initial cultures of Raji cells set at varying cell numbers (105–102). Untransfected Raji cells and g1NANP plasmid served as negative and positive control, respectively. (b) Nested PCR amplification was performed on DNA extracted from 105 Raji cells 24 h after incubation with different amounts (50 mg–0.5 ng) of plasmid. Untransfected Raji cells and g1NANP plasmid served as negative and positive control, respectively. (c) Nested PCR amplification was performed 24 h after spontaneous transgenesis on DNA extracted from 1  106 cells. Untransfected cells and g1NANP plasmid served as negative and positive control, respectively.

43

Expression studies in Burkitt’s lymphoma cells Evidence of internalization together with the notion that the transgene possesses a B-cell-specific promoter prompted us to study specific mRNA transcription in transgenic B cells. Transgenic mRNA synthesis was demonstrated by reverse transcriptase (RT)-PCR in BJAB cells 24 h after spontaneous transfection using as few as 100 cells (Figure 3A). To assess the persistence of expression, studies were performed in cultures of transgenic cells stabilized by selection with G418 Gene Therapy

Transgenesis of human B lymphocytes G Filaci et al

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Table 1

Identification of plasmid DNA by immunoelectron microscopy in human PBL

Experimental conditions

PBL+plasmid PBL

No. of fields

Mean nuclear area (mm2)

Mean cytoplasmic area (mm2)

Extracellular area (mm2)

No. of intracytoplasmic gold particles

No. of extracellular gold particles

24 24

16 995.61 16 510.43

16 319.50 16 126.30

9679.93 9925.27

376 51

0 0

The difference of the number of intracytoplasmic gold particles between PBL incubated with the plasmid and the control. PBL was statistically significant (Po0.001), as calculated by Student’s t-test.

a

c

A

d

B a

1

36%

2

b

3

4

5

6

c

0 1 2 3 4 0 1 2 3 4 100 101 102 103 104 10 10 10 10 10 10 10 10 10 10 EGFP Anti-lgD Anti-lgM

b

e

Figure 2 Immunoelectron microscopy analysis of transgenic human PBL. Analysis was performed on cells fixed after removal of plasmid DNA. (a) Immunoreactive gold particles identify DNA in endosome-like structures (double arrow: 4) and free in the cytoplasm (-); the lower left corner shows part of the nucleus (magnification  78 000). (b) Dual immunolabeling with monoclonal antibody against DNA (10 nm gold particles) (-) or against HLA class II molecules (15 nm gold particles) (double arrow: 4) shows colocalization of DNA and HLA class II molecules in endosomes (E) (magnification  75 000). (c) Immunoreactive DNA is found in endosomes (clear vesicles (left arrow: -) and lysosomes dark vesicles) (right arrow: ’). The nucleus displays diffuse immunoreactivity due to nuclear DNA (lower right corner). (d) Immunoreactive DNA is seen in endosomes (double arrow: 4), free in the cytoplasm and close to the nucleus (N). The nucleus displays diffuse immunoreactivity due to nuclear DNA (lower left corner) (magnification  65 000). (e) Immunoreactive DNA is found free in the cytoplasm close to the nuclear membrane (-) (magnification  78 000).

(1.3 mg/ml) since the backbone pNeog1 plasmid carries the neomycin-resistant gene.26 The cells were analyzed weekly by RT-PCR. In two experiments using BJAB cells, a product was amplified by RT-PCR 1 month after spontaneous transgenesis, but this required subcloning and expansion of transgene-positive cells under G418 Gene Therapy

Figure 3 Expression studies in Burkitt’s lymphoma cells after spontaneous transgenesis. (A) RT-PCR of the transgene mRNA extracted from BJAB cells 24 h after spontaneous transgenesis with plasmid DNA–EGFP. Lane 1, ladder; lane 2, mRNA extracted from 104 cells; lane 3, mRNA extracted from 102 cells; and lane 4, mRNA extracted from 100 cells; lane 5,mRNA extracted from 104 naı¨ve cells (negative control); and lane 6, plasmid DNA (positive control). (B) FACS analysis of magnetically enriched BJAB cells 24 h after spontaneous transgenesis with plasmid DNA-EGFP. (number) indicates the percentage of EGFP-positive cells. After transgenesis cells were also analyzed for surface IgD (panel b) and IgM (panel c) expression. Gray histograms refer to nontransfected BJAB cells (negative control).

selection (not shown). The same result was obtained in six experiments performed with Raji cells. Additional experiments were performed in BJAB cells using a transgene containing the coding region for the enhanced green fluorescent protein (EGFP) to permit visualization and enumeration of transgenic cells by flow cytometry. EGFP was inserted at the end of the constant region gene under control of the Ig promoter and served as an indicator of the Ig transgene expression. To enhance visualization, cells were cotransfected with a plasmid coding for a membrane-bound molecule (murine H-2 Kk). Since a significant portion of the cells internalize and express both plasmids, EGFP-positive cells were enriched using anti-Kk magnetic beads. In 12 out of 13 independent experiments (11 performed with Burkitt’s lymphoma cells and two with EBV-transformed lymphoblastoid cells), EGFP-positive cells were readily visualized (Figure 3B, panel a). By FACS, EGFP-positive cells were also positive for surface IgM and IgD expression (Figure 3B, panels b and c).

Expression studies in human PBL Expression in normal human PBL was first assessed by RT-PCR. In the experiment shown (Figure 4a), we analyzed the mRNA extracted from PBL of three

Transgenesis of human B lymphocytes G Filaci et al

45 1

2

3

4

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6

a

7

b 85%

Anti-CD19

a

89.7%

100 101 102 103 104 EGFP

100 101 102 103 104 EGFP

Number of events

100 101 102 103 104

100 101 102 103 104

79.5%

79%

100 101 102 103 104

100 101 102 103 104

EGFP

EGFP

Figure 4 Expression studies in PBL after spontaneous transgenesis. (a) RT-PCR of the transgene mRNA extracted from 1  104 transgenic PBL 24 h after transgenesis with plasmid DNA-EGFP (lanes 2, 4, and 6). Untransfected (naı¨ve) cells served as specificity control (lanes 1, 3, and 5). Lane 7 – ladder. Data show three experiments representative out of seven independent experiments. (b) FACS analysis of magnetically enriched PBL 24 h after spontaneous transgenesis with plasmid DNA–EGFP. Numbers indicate the percentage of EGFP positive cells. Data show four independent experiments representative of a total of 29 experiments. Gray histograms refer to nontransfected PBL (negative control).

different individuals transfected with plasmid DNA– EGFP. The mRNA extracted from the same PBL before spontaneous transgenesis (naı¨ve PBL) served as a negative control. A clear mRNA product was amplified from all three samples, but not from the naı¨ve cells counterpart. This demonstrates specific transcription after spontaneous transgenesis. Expression of the transgene product was monitored by flow cytometry on Kkenriched PBL. Transgenic lymphocytes could be easily identified as EGFP-positive cells, as exemplified in four independent PBL samples (Figure 4b). Overall, EGFPpositive lymphocytes were easily demonstrable in 29 out of 29 independent experiments proving that spontaneous lymphocyte transgenesis is a highly reproducible event. We calculated that the efficiency of transfection in human PBL, taking into consideration the enrichment factor (ie, the number of times cells were enriched over the original number of cells), is B170.4%.

Transgenic lymphocytes in PBL are B lymphocytes Transcription in B lymphoma cells and PBL, together with the use of a transgene controlled by a B-cell-specific promoter, suggested that B lymphocytes are the target of spontaneous transgenesis. To prove this point, EGFPpositive cells in PBL were stained with a phycoerythrin (PE)-conjugated murine monoclonal antibody against CD19, a marker of human B lymphocytes. In all, 85–90% of detectable EGFP-positive cells were also CD19positive (Figure 5a and b). Since PBL contain 10–15% B lymphocytes, the average efficiency of spontaneous transgenesis in the B lymphocyte population is estimated

Anti-IgD

80%

89%

d Anti-CD27

c b

100 101 102 103 104 EGFP

100 101 102 103 104 EGFP

Figure 5 Phenotypic analysis of PBL after spontaneous transgenesis. (a, b) Expression of CD19 in magnetically enriched (EGFP-positive) PBL from two different donors. PBL used in the experiment correspond to PBL used in Figure 4. (c) Expression of IgD and CD27 (d) in magnetically enriched (EGFP-positive) PBL 24 h after spontaneous transgenesis.

to be B10%. B lymphocytes in PBL are divided into two general categories, naı¨ve and memory, which in humans are distinguished by the membrane expression of CD27.27 An experiment performed to determine which of the two populations was EGFP positive showed that transgenic lymphocytes are IgD-positive/CD27-negative cells (Figure 5c and d). This indicates that naı¨ve mature B lymphocytes are the target population of spontaneous transgenesis.

Spontaneous lymphocyte transgenesis with plasmid DNA under control of the CMV promoter Additional experiments were performed to verify whether the phenomenon of spontaneous internalization and expression was dependent on the type of plasmid and promoter used. We used a commercially available plasmid coding for EGFP under the control of the CMV promoter (pEGFP-N1). In experiments performed in parallel we found no differences between the rates of transfection in BJAB cells (Figure 6a) or human PBL (Figure 6b) transfected either with pEGFP-N1 or with the plasmid with the B-cell promoter. Furthermore, a phenotypic analysis of transgenic PBL (Figure 6c,d) showed that B lymphocytes rendered transgenic for pEGFP-N1 share the same surface markers (IgD þ / CD27) shown in Figure 5. Collectively, these results suggest that spontaneous transgenesis occurs irrespective of the promoter in the plasmid DNA and is thus a general property of B lymphocytes. Spontaneous lymphocyte transgenesis is functional Under normal conditions the signal sequence directs the synthesis of transgenic Ig H chains to the endoplasmic reticulum.28 Then a sizable fraction of newly synthesized polypeptide enters the catabolic pathway, which could be either local29 or proteosome mediated,30 yielding peptides that can load the groove of MHC class I molecules in the endoplasmic reticulum.31 The peptide– MHC class I complex is then exported to the cell surface serving as target recognition for specific CD8 þ T lymphocytes. To test if these events would occur after Gene Therapy

Transgenesis of human B lymphocytes G Filaci et al

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104

100

100 104 100

100 101 102 103 104 EGFP

BJAB

100 101 102 103 104 PBLs

100 101 102 103 104

100

IgD

104

100 101 102 103 104 EGFP

c

γ1-EGFP

104

Number of Events

b

p-EGFP-N1

100

Number of Events

a

100

CD27

d

100 101 102 103 104

104

100 101 102 103 104 EGFP

100 101 102 103 104 EGFP

100 101 102 103 104

Figure 6 Spontaneous transgenesis with plasmid DNA under control of a CMV promoter. (a) FACS analysis of BJAB cells transfected with plasmid DNA under control of a CMV promoter (pEGFP-N1) and in parallel with a g1-EGFP plasmid. The experiment was repeated twice with similar results. (b) FACS analysis of human PBL transfected with pEGFP-N1 and in parallel with g1-EGFP plasmids. The experiment was repeated four times with similar results. (c–d) Phenotypic analysis of PBL after spontaneous transgenesis with pEGFP-N1 and in parallel with g1-EGFP plasmids. Expression of IgD (panel c) and CD27 (panel d) in cells after transgenesis.

spontaneous transgenesis, HLA-A2 þ and Ig-synthesizing BJAB cells were incubated with plasmid DNA whose Ig transgene V region had been engineered with an HLAA2-restricted epitope from the melanoma-associated MART-1 antigen (MART-127–35).23 Transgenesis was monitored by PCR (not shown) and FACS analysis. To generate a homogeneous target population, cells were placed under G418 selection and subcloned to enrich for EGFP-positive cells. Wells with visible growth were checked by FACS for EGFP expression (Figure 7, upper row). EGFP-positive cultures were then used as targets for two human HLA-A2-restricted/MART-127–35-specific cytotoxic T lymphocyte (CTL) clones in a conventional 51 Cr release assay. In three independent experiments, transgenic BJAB cells were lysed by two effector CTL clones23 (Figure 7, lower rows) with a maximal lysis at an effector:target ratio of 25:1 of 74 and 46%, respectively. No lysis was observed using nontransfected BJAB cells as targets. To further ensure that lysis was specific, an additional experiment was performed where BJAB cells Gene Therapy

transfected with a control plasmid (without the MART-1 antigen) were used as target together with transgenic BJAB cells (clone 17). No lysis was observed at any of the E:T ratios used (Figure 8). Therefore, lysis of transgenic cells requires expression, processing, and presentation of the MART-127–35 peptide.

Discussion We demonstrated that a brief contact in vitro between plasmid DNA and B lymphocytes leads to DNA uptake and cell transgenesis. Based on the data presented, spontaneous transgenesis appears to be a constitutive characteristic of B lymphocytes. Remarkably, spontaneous transgenesis was reproducibly observed by different techniques (PCR, RT-PCR, and FACS) in 100% of the PBL-tested samples (29 samples including four independent replicates). In spite of the ease of reproducibility, the exact mechanism of DNA internalization can only be speculated. Studies on spontaneous DNA internalization in vitro (using viral DNA or oligonucleotides)25,32,33 or in vivo (using plasmid DNA)34 exclude the fact that DNA molecules penetrate through pores or areas of partial disruption of the membrane. Thus, two possibilities are considered. One is internalization by pinocytosis (including fluid-phase endocytosis and macropinocytosis). The other is internalization by receptor-mediated endocytosis. Somatic cells have been reported to express a variety of DNA receptor candidates,25,32,33,35–38 none of which showed to be uniquely essential for DNA uptake. Specifically, receptors mediating DNA internalization in human B lymphocytes are poorly understood. Early studies identified a 28–34 kDa molecule that internalizes high molecular weight DNA with a kinetics typical of receptor-mediated endocytosis.39,40 More recently, a B130 kDa membrane-associated protein with a RING finger domain has also been described with ubiquitous distribution including lymphoid organs.41 Toll receptor 9 (TLR-9), a transmembrane protein localized at ‘endosome-like’ cytoplasmatic structures,42–44 mediates the activatory-intracellular signaling of CpG-containing oligonucleotides via interaction with an intracellular MyD88 adapter molecule, IRAK, and TRAF645 but not their internalization. Lastly, HLA-class II molecules that are constitutively expressed on B lymphocytes and undergo rapid internalization and recycling upon ligation46–48 could be at play based on the observation that HLA-DR molecules bind oligonucleotides with high affinity (Kd 297.9–116.7 nM).49 Although attractive, our evidence in favor of this hypothesis is modest. For instance, DNA internalization was diminished but not abrogated in HLA class II competent (Raji) cells as compared to HLA class II deficient (RJ 2.2.1) cells (not shown). By analogy, EGFP expression in B lymphocytes from MHC class II knockout mice was reduced (B40%) compared with that of wild-type lymphocytes (not shown). Collectively, it appears that MHC class II molecules may contribute in part to DNA internalization, but overall the process is multifactorial and largely unknown. Our data show the presence of internalized DNA in the endosomal, lysosomal, and cytoplasmic compartments. While the data imply that the endocytosed DNA diffuses out of the late endosomes, an event known to

Transgenesis of human B lymphocytes G Filaci et al 100

100

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Cell count 0

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100 101 102 103

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EGFP CTL #1088

T2 100

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60

40

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40

20

20

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20

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0

0

0

0

0

Percent lysis

100

BJAB

Percent lysis

CTL #1235

Clone #11

Clone #12

Clone #15

Clone #19

+ + MART-1 Peptide

Figure 7 Transgenic B lymphocytes process and present the AAGIGILVT peptide to specific CTL. BJAB (HLA-A2 þ ) Burkitt’s lymphoma cells were rendered transgenic with plasmid g1MART–EGFP, which codes for the melanoma-associated MART-1 peptide (27AAGIGILVT35) in the CDR3 of the variable region of the transgene H chain. Transgenic BJAB cells were enriched by two rounds of subcloning and selection with G418. The experiment shown was performed with four clones at the time when expression of the g1MART transgene (EGFP positivity) was assessed by FACS (upper row). Lysis of g1MART transgenic cells by CTL clone #1088 (middle row) and #1235 (lower row) 23specific for the MART-127–35 peptide is shown. TAP-deficient/HLAA2 þ T2 (white bars) and BJAB (gray bars) cells, with or without MART-127–35 peptide, served as positive and negative target control, respectively. Values refer to percent lysis at a 25:1 E:T ratio. Tests were performed in triplicate and on the same day to avoid intertest variations. Transgenic BJAB clones were used 48 h after FACS analysis. The experiment is representative of three independent experiments.

TIL #1088

TIL #1235

75

T2 T2 + peptide clone 17 BJAB+control DNA

50 25 0

100 Lysis Percent

Lysis Percent

100

75 50 25 0

50:1 12:1 3:1 E:T Ratio

50:1 12:1 3:1 E:T Ratio

Figure 8 Specificity of the processing by transgenic B lymphocytes to CTL. BJAB cells were rendered transgenic with plasmid g1MART–EGFP (clone 17) or with a control plasmid DNA without the MART antigen. Lysis of g1MART transgenic cells by CTL clone #1235 and #1088 is shown. T2 cells with or without MART-127–35 peptide, served as positive and negative target control, respectively. Values refer to percent lysis at different E:T ratios. Tests were performed in triplicate.

depend on specific signal sequences in the endocytic receptor(s),50,51 the mechanism(s) allowing the diffusion out of the endosome in our case are not known. Classically, ligands internalized through endocytic re-

ceptors localize first to the early endosome and subsequently to the late endosome/lysosome compartment. Occasionally, however, specific signal sequences sort endocytosed material to the late endosome. Examples of this type are DEC-205,52 an endocytic receptor expressed on murine dendritic cells and B lymphocytes,53 and DCSIGN, a C-type lectin receptor present only on dendritic cells.54 Interestingly, human B lymphocytes express a DEC-205 homologue (the gp200-MR6 molecule),55 which possesses the same GFSSVRY sorting sequence as DEC205.52 Which of these eventualities applies to our model remains unknown and deserves future study. Exogenous DNA was seen (Figures 3 and 4) to reach the nucleus consistent with the demonstration of transcription. Does the transgene remain episomal or integrate into chromosomal DNA? Studies on Burkitt’s lymphoma cells show that protracted expression of the transgene requires continuous selection by G418, arguing against integration. The observed instability of the transgene in B lymphocytes after spontaneous transgenesis in the absence of selective pressure is consistent with an extrachromosomal localization of the plasmid DNA56,57 so that at each round of mitosis only one of the two daughter cells inherits the transgene causing its Gene Therapy

Transgenesis of human B lymphocytes G Filaci et al

48

rapid dilution. Theoretical considerations also argue that integration is a remote eventuality. It is estimated that integration of foreign genes depends on the cell type and the input molecules of exogenous DNA.58 In the case of spontaneous transgenesis, we know that (1) the input DNA is 104 molecules per lymphocyte and (2) lymphocytes need not be activated and do not enter the cell cycle and proliferate after DNA uptake (unpublished data). Thus, assuming an estimated maximal probability of integration-mediated activation of an oncogene or inactivation of a tumor suppressor gene in the order of 1016,58 the maximum cumulative probability of having harmful effects after spontaneous lymphocytes transgenesis would be in the order of 1012. Our studies were designed to favor the expression of a reference transgene and enable detection of downstream functional events. Indeed, in PBL mRNA transcription and expression of the Ig transgene–EGFP fusion protein were always observed. Importantly, we found that transgenic B cells process and present the transgene product to MHC-restricted CTL, demonstrating that spontaneous transgenesis includes downstream functions that impart B lymphocytes with new immunological properties. B cells are quite capable to process and present antigen once this has been efficiently internalized via the B-cell antigen receptor (BCR).59 However, this requires sufficient frequencies of B cells with highaffinity BCR, conditions realized after re-encounter with antigen but virtually impossible for naı¨ve B lymphocytes. Our experiments show that spontaneous transgenesis is a way of programming the intracellular synthesis of antigen in naı¨ve B lymphocytes bypassing the BCR. The existence of a ‘program’ for DNA internalization and intracellular trafficking in human B lymphocytes, together with the fact that spontaneous lymphocyte transgenesis is equally effective with mouse or macaque lymphocytes (our unpublished data), begs the question ‘IS there an evolutionary explanation to spontaneous transgenesis of B lymphocytes?’ In our opinion this is an unexpected byproduct of the antigen processing/presenting pathway since DNA internalization yielded a functionally relevant and seemingly surprising downstream effect: processing and presentation of the transgene product along the MHC class I pathway (Figure 7). This is reminiscent of the way exogenous antigens internalized by receptor-mediated endocytosis are presented by MHC class I molecules60 in macrophages61,62 and in dendritic cells.63 This type of antigen processing/ presentation requires that the endocytosed antigen be released from the endosome/lysosome compartment into the cytosol for processing by the proteasome, implying the existence of a mechanism for facilitated transmembrane transport from the lumen of endosomes into the cytosol.64 By analogy, in B lymphocytes the internalized DNA also diffuses out of the late endosomes free to reach the nucleus and undergo transcription. This conserved ‘program’ reflects a general, and possibly ancestral, property of lymphomyeloid cells not previously recognized in B lymphocytes. In conclusion, we demonstrated spontaneous transgenesis of B lymphocytes by plasmid DNA and showed that this event imparts B lymphocytes with new functional properties. Although spontaneous transgenesis of cultured cells with genomic DNA has been known for several decades13,16 and B lymphocytes have been

Gene Therapy

transduced using retroviral vectors,65,66 to our knowledge this is the first report showing functional transgenesis of primary human B lymphocytes in the absence of any extrinsic cell manipulation and activation using a nonviral vector. As shown, spontaneous transgenesis is a simple way to program ad hoc the synthetic machinery of a B lymphocyte and jump start processing and presentation of peptides expressed by the transgene product. Since naı¨ve B lymphocytes undergoing spontaneous transgenesis are turned into bona fide antigen-presenting cells, the phenomenon disclosed here is relevant to somatic cell/gene therapy approaches using transgenic B lymphocytes to program adaptive immune responses in vivo.

Experimental procedures Cells BJAB and Raji Burkitt’s lymphoma cells were obtained through the courtesy of Dr D Mosier (The Scripps Research Institute, La Jolla, CA, USA) and Dr R Accolla (University of Insubria, Varese, Italy). The expression of the HLA-A2 allele product on BJAB cells was confirmed by FACS analysis using the anti-HLA-A2 monoclonal antibody BB7.2.67 EBV-transformed lymphoblastoid cell lines were kindly provided by Dr R Accolla. PBL were obtained from heparinized peripheral blood of healthy donors by centrifugation on the Ficoll gradient (Sigma, St Louis, MO, USA). Procedures were covered by an Institutional Review Board-approved protocol. Plasmids Plasmid g1NANP carries a chimeric H chain gene in which the productively rearranged murine VH62 region gene26 is joined to the human g1 C region gene of the pNeog1 vector.68 The V region gene was modified in the third CDR3 by introduction of three NANP repeats.69 The promoter and enhancer elements in this plasmid are those constitutively existing in Ig H chain genes, and were described previously.26 Plasmid g1MART carries the 27 AAGIGILTV35 peptide of the MART-1 melanomaassociated antigen23 in the CDR3. To allow for detection of transgenic cells by FACS, the gene for EGFP was inserted at the C terminus of the g1 constant region of both plasmids. Plasmid pEGFP-N1 was purchased from BD Biosciences Clontech (Palo Alto, CA, USA) (catalog # 6085-1). Spontaneous transgenesis Cells were washed with PBS without Ca2 þ and Mg2 þ (Cellgro, Hendon, VA, USA) to remove residual fetal calf serum (FCS). Cells (1–4  106) were resuspended in 200 ml of PBS without Ca2 þ and Mg2 þ and incubated with 25 mg of plasmid DNA for 1 h at 371C. During the incubation the cells were gently shaken every 15 min. The cells were then washed three times with PBS, resuspended in culture medium (RPMI-1640) supplemented with HEPES buffer, glutamine, 10% FCS at 371C in 5% CO2 atmosphere overnight. In some experiments the number of cells varied from 102 to 106 and the amount of plasmid DNA per 105 cells ranged from 500 pg to 50 mg.

Transgenesis of human B lymphocytes G Filaci et al

PCR and RT-PCR DNA was extracted using the QIAamp (Qiagen, Valencia, CA, USA) and quantitated on a 1% agarose gel. PCR was performed with a total of four sets of primers (pCL/ pCD, pNEL/pNED, pDETup/pNED, and pbA1/pbA2). pCL (50 -TTATTGAGAATAGAGGACATCTG-30 ) and pCD (50 -ATGCTCATAAAACTCCATAAC-30 ) were used to amplify a fragment of 566 bp corresponding to the whole VDJ region of the transgene.70 The nested PCR was performed using pNEL (50 -AGCACCTACTATCCA GACACT-30 ) and pNED (50 -GTAGTCCATACCATGA GAGTA-30 ) amplifying a fragment of 200 bp and and pDETup (50 -GCCAGACTCCAGAGAAGA-30 ) pNED amplifying a fragment of 256 bp. Primers pbA1 (50 -TGGGCCGCCCTAGTCACC-30 ) and pbA2 (50 -CGT TTGGCCTTAGGGTTCAG-30 ) were used to amplify the b-actin gene, a control for DNA extraction, while establishing the experimental conditions. The first round of PCR consisted of 30 cycles at 941C for 45 s, 581C for 45 s, and 721C for 45 s, using 0.2 mM of each primer, 0.1 mM of each deoxynucleotide, 1.5 mM MgCl2 in 20 mM Tris-HCl (pH 8.4)/50 mM KCl, and 1 U of Taq DNA polymerase (Gibco). Nested PCR consisted of 25 cycles of amplification performed with the same protocol used for the first round PCR. Total RNA was extracted by RNeasy Mini Kit (Qiagen). cDNA was synthesized using primer PCD and Superscript IIt Reverse Transcriptase (Gibco). Annealing and reverse transcription were performed at 651C for 10 min and at 421C for 50 min, respectively. PCR amplification of the obtained cDNA was performed as previously described. cDNA amplification was checked by electrophoresis using 1.5% Agarose 1000 (Gibco BRL). Immunoelectron microscopy PBL were processed for immunoelectron microscopy after 60 min of incubation with plasmid DNA. Briefly, cells were washed and fixed with a solution of 2% paraformaldehyde and either 0.5 or 2.5% glutaraldehyde in 0.1 M cacodylate buffer (pH 7.4) for 1 h in an ice bath. Cells were then resuspended in cacodylate buffer and centrifuged at 300 g for 5 min. The pellets were embedded either in an Epon-Araldite resin mixture or in hydrophilic London White Resin (LWR medium grade, London Resin Company, Basingstoke, UK). Ultrathin sections were processed for immunocytochemistry, counterstained with uranyl acetate and lead citrate, and examined by electron microscopy. PBL not incubated with plasmid DNA served as negative controls. A colloidal gold-labeling technique71 was used to localize DNA and HLA class II molecules. Briefly, ultrathin sections of resin-embedded material were collected on 300 mesh nickel grids, placed on a drop of 10% nonimmune goat serum for 1 h at room temperature to prevent nonspecific binding of Ig, and incubated overnight at 41C with an anti-DNA IgM (BoehringerMannheim, Indianapolis, IN, USA) monoclonal antibody diluted in 0.5 M Tris-HCI buffer (pH 7.3) containing 1% bovine serum albumin and 0.5% sodium azide (TBS). All grids were then rinsed three times in TBS, incubated for 1 h at room temperature with goat anti-mouse IgM conjugated with 10 nm gold particles (EM GAMM 10, British BioCell, Cardiff, UK), washed three times for 30 min in TBS, washed again in distilled water, and

contrasted with uranyl acetate and lead citrate. Control sections were similarly processed except that the antiDNA antibody was omitted or replaced with nonimmune mouse serum or monoclonal antibody BD-5 (IgM) of unrelated specificity.72 Colocalization of DNA and HLA class II molecules was studied by immunostaining using, in addition, an anti-HLA class II IgG (LGII-612.14) monoclonal antibody.73 The grids were then rinsed in TBS, incubated for 1 h at room temperature with goat anti-mouse IgG conjugated with 15 nm gold particles (EM GAM 15, British BioCell, Cardiff, UK), washed three times for 30 min in TBS, washed again in distilled water, and counterstained with uranyl acetate and lead citrate. All samples were examined with a Zeiss EM902 electron microscope (Oberkochen, Germany). The morphometric analysis was performed as follows. Immunogold particles were counted in the cytoplasms of 24 randomly selected anti-DNA-stained PBL, which had been previously incubated with the g1NANP plasmid, and in 24 control cells. The number of gold particles in the cytoplasm was assessed by examining 16 319 and 16 126 nm2 of cytoplasmic area in transgenic and control cells, respectively. Scion Image (Beta 4.02 for Windows) image-analysis software (Scion Corporation, USA) was used to calculate the extent of cytoplasmic areas on scanned electron micrographs.

49

Magnetic labeling and separation of transgenic cells Spontaneous transgenesis was performed by incubating cells with plasmid–EGFP together with the PMACS Kk plasmid (Miltenyi Biotec, Auburn, CA, USA) coding for a truncated mouse H-2 Kk molecule as a selectable cell surface marker. This plasmid is part of the MACSelect Kk kit, which provides for the magnetic isolation of stable and transiently transfected mammalian cells. Briefly, 1–4  106 cells were incubated for 60 min at 371C with 25 mg of the plasmid–EGFP and 5–10 mg of PMACS magnetic plasmid in a total of 200 ml of sterile PBS without Ca2 þ and Mg2 þ . The cells were then washed and incubated overnight in RPMI-1640 containing 10% FCS at 371C in 5% CO2 atmosphere. The cells were again harvested and resuspended in 320 ml PBS/0.5% BSA/ 5 mM EDTA (PBE) containing 80 ml of MACSselect Kk microbeads. The suspension was incubated for 15 min at 6–121C. Transgenic and magnetically labeled cells were enriched by positive selection on a column MS þ /RS þ mounted on the magnetic field of a MACS separator. Negative cells were collected in the flowthrough. After washing, the column was removed from the separator and transgenic cells were flushed out with PBE using a plunger. FACS analysis Magnetically enriched cells and cells cultured in G418 selection after spontaneous transgenesis with the plasmid–EGFP were analyzed by FACS using cells transfected with only the magnetic plasmid or untransfected cells as a negative control. A gate in the forward versus side scatter map was set to exclude debris and dead cells. In dual staining experiments, magnetically enriched cells were stained with PE-conjugated monoclonal antibodies to human CD19 (clone HIB19), CD27 (clone M-T271), anti-IgM (clone G20-127), and anti-IgD (clone IA6-2) (Pharmingen, San Diego, CA, USA) by incubation for 45 min at 41C. After washings, cells were resuspended in Gene Therapy

Transgenesis of human B lymphocytes G Filaci et al

50

300 ml of PBS and analyzed in a FACsCalibur (Beckton & Dickinson, CA, USA). 51

Cr-release assay

EGFP þ BJAB cells expressing g1MART were generated by spontaneous transgenesis followed by culture under continuous G418 selection. After subcloning, positive cultures were identified by FACS analysis and used as a target in a conventional 51Cr-release assay where the HLA-A2-restricted MART-127–35 specific CTL, TIL #1235, and TIL #108823 (the kind gift of Dr N Restifo, National Cancer Institute, Bethesda, MD, USA) served as the effectors per our standard assay conditions.74 The percentage of lysis was calculated as follows: 100  (sample cpmspontaneous release cpm)/(maximum releasespontaneous release).

Acknowledgements We are thankful to Drs MG Farquhar, W Loomis, and J Urbain for critical reading of the manuscript and their helpful suggestions. We also thank G Almanza and I LacKamp (Cosmo Bioscience) for their help in the performance of PCR work and A Necchi (University of Pavia) for assistance with the electron microscopy. This work was supported in part by a grant from the School of Medicine of the University of California, San Diego, and NIH Grants RO1CA77427 and R21AI49771 (to MZ). GF was a recipient of a short-term mobility grant from Consiglio Nazionale delle Ricerche and from the Compagnia di SanPaolo (Italy).

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Gene Therapy

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