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lentiviral vectors pseudotyped with measles virus glycoproteins. M Schoenhals1, C ... able to stably introduce a genetic modification into malignant PCs to get a ...
Leukemia (2012) 26, 1663 - 1670 & 2012 Macmillan Publishers Limited All rights reserved 0887-6924/12 www.nature.com/leu

ORIGINAL ARTICLE

Efficient transduction of healthy and malignant plasma cells by lentiviral vectors pseudotyped with measles virus glycoproteins M Schoenhals1, C Frecha2,3,4, A Bruyer1, A Caraux1, JL Veyrune5, M Jourdan1, J Moreaux1,5, F-L Cosset2,3,4, E Verhoeyen2,3,4 and B Klein1,5,6 A lot of genes deregulated in malignant plasma cells (PCs) involved in multiple myeloma have been reported these last years. The expression of some of these genes is associated with poor survival. A critical step is to elucidate the biological mechanisms triggered by these gene products. Such studies are hampered by the difficulty to obtain malignant PCs and to genetically modify them. Usual lentiviral vectors (LVs) pseudotyped with vesicular stomatitis virus envelope glycoprotein poorly transduced healthy and malignant PCs. Here, we report that LVs pseudotyped with the hemagglutinin and fusion glycoproteins from the measles Edmonston strain (H/F-LVs) can efficiently and stably transduce healthy and primary malignant PCs, without modifying their main phenotypic characteristics. Both LV pseudotypes efficiently transduced human myeloma cell lines. Importantly, both healthy and malignant PCs expressed CD46 and SLAMF1/CD150 membrane proteins, which are critical receptors for binding and productive genetic modification by H/F-LVs. The ability to efficiently introduce and express a given gene into PCs opens the possibility to study in detail PC biology. Leukemia (2012) 26, 1663 -- 1670; doi:10.1038/leu.2012.36 Keywords: plasma cell; transduction; multiple myeloma; measles glycoproteins

INTRODUCTION Multiple myeloma (MM) is characterized by the accumulation of malignant plasma cells (PCs), primarily in the bone marrow.1 Normal PCs are rare cells in the body, located mainly in the bone marrow (0.5% of bone marrow cells) and mucosa. These normal PCs are quiescent cells in vivo and may survive for years, insuring long-term humoral memory.2 MM disease is preceded by a several year-lasting premalignant monoclonal gammopathy with undetermined significance (MGUS).3 MGUS may transform to MM with a 1% rate per year, independent on MGUS duration.4 MM disease is heterogeneous in terms of molecular abnormalities in malignant PCs,5 and the use of high throughput methodologies has revealed many genes whose copy number or expression is associated with a difference in patients’ treatment response or survival. But the study in depth of the biological role played by deregulated gene products is hampered by the availability of primary healthy or malignant PCs, the difficulty to culture and/or to genetically modify them. This is particularly the case when no tools (recombinant protein or inhibitor) exist to study the function of a gene product. This explains why most biological studies use human myeloma cell lines (HMCLs), which are obtained form a minority of patients with a special MM disease entity, that is, patients with extramedullary proliferation.6 The biological concepts obtained with these HMCLs could be limited to this minority of patients, in whom malignant PCs are no more or less dependant on the bone marrow environment, likely due to additional deregulations of survival and cell cycle.7 To further progress in the understanding of MM disease, it is critical to be able to stably introduce a genetic modification into malignant PCs to get a forced expression or knockdown of a gene. It is also 1

important to manipulate PCs, to investigate whether myeloma genes can modify the biology of healthy PCs and transform them into premalignant or malignant PCs. Whereas vesicular stomatitis virus G (VSV-G) pseudotyped lentiviral vectors (LVs) can transduce HMCLs,8 they poorly transduce primary malignant PCs, which are weakly cell cycling. We have shown recently that LVs incorporating the Edmonston strain measles virus (MV) with hemagglutinin and fusion glycoproteins (H/F-LVs) can efficiently and stably transduce quiescent human B lymphocytes9 or T lymphocytes.10 The MV H glycoprotein binds to signaling lymphocyte activation molecule (SLAMF1/CD150) and to CD46, which are expressed on a subpopulation of quiescent B or T lymphocytes. Transduction of malignant PCs by H/F-LVs should be possible as several reports have documented the ability of oncolytic MV to efficiently infect and kill primary malignant PCs or HMCLs, in particular because malignant PCs express CD46, and this strategy was proposed as a therapeutic option to kill malignant PCs.11 - 13 In this study, we report that H/F-LVs can efficiently transduce primary malignant PCs as well as healthy PCs, where VSV-G-LVs failed. VSV-G-LVs can transduce HMCLs but with a lower efficacy than MV-LVs. MATERIALS AND METHODS Cell samples Bone marrow cells were obtained from 10 patients with MM for routine clinical evaluation of the disease. Excess cells were used after obtaining patients’ written informed consent in accordance with the Declaration of Helsinki and agreement of the Center for Biological Resources of

INSERM U1040, Institute for Research in Biotherapy, CHU Montpellier, Hospital St Eloi, Montpellier, France; 2INSERM, U758, Human Virology Department, EVIR, Lyon, France; Ecole Normale Supe´rieure de Lyon, Lyon, France; 4Universite´ de Lyon, UCB-Lyon1, Lyon, France; 5CHU Montpellier, Institute of Research in Biotherapy, Montpellier, France and 6 Universite´ MONTPELLIER1, UFR Me´decine, Montpellier, France. Correspondence: Professor B Klein, INSERM U1040, Institute for Research in Biotherapy, CHU Montpellier, Hospital St Eloi , Av Augustin Fliche, Montpellier 34295, France. E-mail: [email protected] Received 19 September 2011; revised 28 December 2011; accepted 25 January 2012; accepted article preview online 9 February 2012; advance online publication, 2 March 2012 3

Plasma cell transduction with measles lentiviruses M Schoenhals et al

1664 fluorochromes. Isotype control was used to evaluate the level of background fluorescence on PC populations. Data acquisition was performed with a Cyan flow cytometer driven by Summit 4.3 software (Beckman Coulter) or a FACScan cytometer (BD). Data were analyzed with the Kaluza 1.1 software (Beckman Coulter) or CellQuest (BD). Cell debris and doublets were excluded in the first step. PCs were defined as CD138 þ cells.

Montpellier University Hospital (N1DC-2008-417). Cell cycle profiles of primary malignant PCs were determined using PI staining and the algorithm ModFit was used to assign the different cell cycle phases.14 For gene expression data, we used Affymetrix U133 plus 2.0 publicly available data of our cohort of 206 newly-diagnosed patients treated with induction treatment and then high dose melphalan and autologous stem cell transplantation (http://www.ebi.ac.uk/arrayexpress, under accession number E-MTAB-362).15 HMCLs were obtained and characterized in our laboratory.16 - 18 Healthy plasmablasts or PCs were obtained using our three-step in vitro model making it possible to obtain six syndecan-1 þ PCs starting from one circulating memory B cell within 10 days.19 This threestep model mimics the activation and differentiation processes of B cells mediated at least by T cells and follicular dendritic cells occurring in germinal centers. Step 1 uses a stimulation by CD40L, CpG oligonucleotide activating Toll-like receptor and T-cell cytokines IL-2, IL-10 IL-15; step 2 a removal of CD40L and Toll-like receptor stimulation to promote plasmablastic differentiation and addition of STAT3 activating cytokines; and step 3 an activation of interferon signaling pathway to promote further PC differentiation.

A total of 5  104 cells were deposited onto poly-L-lysine-coated slides, fixed in 4% paraformaldehyde and permeabilized with 0.1% Triton. Non-specific staining was blocked by incubating the slides in 5% BSA-PBS, after which the slides were subjected to labelling with the anti-CD138 mAb (Beckman Coulter, clone MI-15) for 1 h in a moist chamber. Slides were then washed with PBS, incubated for 1 h with an Alexafluor 546-conjugated rabbit anti-mouse antibody (Invitrogen, Molecular Probes, Cergy Pontoise, France) in a dark moist chamber. Slides were washed and mounted with Vectashield containing 40 -6-Diamidino-2-phenylindole (DAPI) (Sigma Aldrich, St Louis, MO, USA), and examined under an epifluorescence Microscope (Leica Microsystems, Wetzlar, Germany).

Plasmids

Culture of transduced cells

H and F glycoproteins are from the Edmonston vaccine strain. They were inserted into pCG plasmids under the control of the cytomegalovirus early promoter. Cytoplasmic tails of all H and F proteins were deleted by truncation of 24- and 30-aa, respectively. The pCMV-G plasmid encoding the VSV-G glycoprotein was described previously.20

Transduced HMCLs were cultured for 1 month with change of the culture medium every 3 - 4 days. Cells were harvested every 7 days for assaying the GFP expression by FACS analysis. Bone marrow mononuclear cells from three patients with MM were transduced with H/F-LVs for 2 days. Cells were washed and malignant PCs together with their bone marrow environment were cultured for 5 days in 200 ml of RPMI1640 culture medium, 10% FCS and IL-6 (2 ng/ml), and the GFP expression was assayed by FACS analysis. The 5  105 in-vitro generated healthy PCs harvested at day 7 of culture were transduced with H/F-LVs and cultured for 3 days on 104 bone marrow stromal cells generated as indicated23 in 1 ml of RPMI1640 culture medium, 10% FCS, IL-6 (10 ng/ml) and IFNa (500 U/ml), washed and cultured for 11 days on 104 bone marrow stromal cells in 1 ml of RPMI1640 culture medium, 10% FCS and IL-6 (10 ng/ml). The GFP expression was assayed at day 3 and day 14 after H/F-LV transduction by FACS analysis.

Lentiviral production Self-inactivating HIV-1-derived vectors were generated by transient transfection of 293T cells in DMEM medium (Invitrogen, Carlsbad, NM, USA) as described.10 Briefly, for VSV-G pseudotyping 3 mg of the pCMV-G plasmid was transfected and for co-display of the different H and F proteins, 3 mg of each envelope plasmid was transfected together with a gagpol packaging plasmid and a plasmid encoding a LV expressing GFP (SIN-HIVSFFVGFP). Virus was concentrated using overnight low-speed (3000 g at 4 1C) centrifugation of viral supernatant and frozen at 80 1C until use. The transduction efficiency and infectious titers were assayed adding to 293T cells serial concentrations of vector preparation and monitoring GFP þ cells by immunofluorescence. Infectious titers were expressed as 293T transducing units per milliliter (TU/ml) as described.21

Transduction of healthy and malignant PCs Cells were cultured in RPMI 1640 medium (Invitrogen, Gibco BRL, Cergy Pontoise, France) supplemented with 10% FCS (PAA Laboratory GmbH, Pasching, Austria) and with 2 ng/ml of IL-6 (Abcys SA, Paris, France) for malignant PCs and HMCLs and in the appropriate step culture conditions for healthy in vitro generated PCs.19 A 2  105 cells were seeded per 48well and were transduced with a multiplicity of infection ranging from 0.125 to 1.5 for HMCLs and up to 7.5 as indicated for malignant or healthy PCs. The percentage of GFP þ cells was determined 72 h after transduction by fluorescence-activated cell sorting (FACS) using a FACScan cytometer (Becton Dickinson (BD), San Jose, CA, USA).

Flow cytometry immunophenotyping Multiparameter analysis was done with antibodies (mAbs) conjugated to fluorescein (FITC), phycoerythrin (PE), PE-cyanin-7 (PE-Cy7), allophycocyanin (APC) and APC-Alexa 750. Specific mAbs for human CD19 (clone J3119), CD20 (clone B9E9), CD38 (clone LS198-4-3), CD45 (clone J-33) and CD138 (clone B-A38) were purchased from Beckman Coulter (Fullerton, CA, USA). CD38 (clone HIT2) and CD56 (N-CAM, clone B159) from BD, and CD46 (clone 8E2), CD150 (clone A12(7D4)) and CD200 (clone OX104) from eBioscience (San Diego, CA, USA). Isotype-matched mouse mAbs recognizing no human antigen were used as controls. Flow cytometry immunophenotyping was performed using a multiparameter immunofluorescence technique as indicated.22 Bone marrow cells, PCs obtained in vitro and HMCLs were labeled with mAbs conjugated to various Leukemia (2012) 1663 - 1670

Immunofluorescence staining

Statistical methods Student’s t-test (nX5) was used to evaluate the statistical significance of differences observed between groups for paired and unpaired variables. P values p0.05 were considered to be associated with statistical significance.

RESULTS Expression of CD46 and SLAMF1/CD150 by healthy and malignant PCs Recently, it was reported that the surface expression of SLAMF1/ CD150 and CD46 MV receptors are absolutely required for primary human lymphocyte transduction.24 Therefore, we evaluated the presence of both receptors on malignant and healthy PCs. As illustrated for one representative patient in Figure 1a, primary malignant PCs expressed CD46 and SLAMF1/CD150 and this was confirmed for 10 other patients with MM (Figure 1b). Primary malignant PCs were poorly cell cycling (1.5% of myeloma cells in the S phase of the cell cycle, range 0.8 -- 3%) (Supplementary Figure S1). Using a large series of newly diagnosed patients with MM, CD46 and SLAMF1/CD150 genes were expressed (present Affymetrix call) in purified malignant PCs of all patients (Supplementary Figure S1). Healthy plasmablasts and PCs express both CD46 and SLAMF1/CD150 genes and proteins (Figures 1a and b and Supplementary Figure S1). Of note, HMCLs did not express SLAMF1/CD150 but expressed CD46 (Figures 1a and b and Supplementary Figure S1). Efficient transduction of primary myeloma cells and normal PCs by measles-virus glycoproteins pseudotyped LVs (H/F-LVs) Given the fact that as well healthy as malignant PCs expressed SLAMF1 and CD46, the two MV receptors, we wanted to verify if & 2012 Macmillan Publishers Limited

Plasma cell transduction with measles lentiviruses M Schoenhals et al

1665 Malignant PCs

Healthy PCs

Healthy Plasmablasts

Mean fluorescence intensity

HMCLs

500

CD46

400

CD150

300 200 100 0 Multiple Myeloma

Healthy PCs

Healthy Plasmablasts

HMCLs

Figure 1. Malignant PCs, healthy PCs and plasmablasts express CD46 and CD150. (a) Cells were labeled with either FITC-conjugated anti-CD46 and PE-conjugated anti-CD138 mAbs or PE-conjugated anti-CD150 and FITC-conjugated anti-CD138 mAbs. Presented are the percentage and mean fluorescence intensity (MFI) of CD46 and CD150 for the gated CD138 þ primary malignant plasma cells, healthy plasmablasts or plasma cells or U266 myeloma cells. (b) Bar plots indicate mean fluorescence intensities±s.d. of surface CD46 and CD150 expression on five samples of either primary malignant PCs, or healthy plasmablasts or PCs, or ten HMCLs.

these cells allowed efficient H/F-LV-mediated transduction. H/FLVs were far more efficient to transduce primary malignant PCs than VSV-G-LVs. Data for a representative patient are shown in Figures 2a and c and mean values for 10 patients in Figure 2b. Using the same multiplicity of infection of 5, 21% of primary malignant PCs (range 9 -- 46%) were transduced with the H/F-LVs with a mean fluorescence intensity in transduced cells of 180 versus 5% transduced malignant PCs with a mean fluorescence of 20 for VSV-G-LVs (P ¼ 0.001). Both H/F-LVs and VSV-G-LVs could efficiently and similarly transduce HMCLs (results not shown). Malignant PCs highly express CD138 and CD38, aberrantly CD56, CD200 or CD117 and did not express CD19 and CD45.25 Data on Figure 3a show these phenotypic characteristics were not modified by H/F-LV transduction. Indeed GFP þ and GFP primary malignant PCs from one representative patient displayed similar mean fluorescence intensity for CD138, CD38 and CD200, and lacked CD19 and CD45. A similar phenotypic stability of H/F-LVtransduced primary malignant PCs was found for two other patients’ samples. The same holds true for HMCLs, whose phenotype was not affected by H/F-LV transduction (Table 1). Transduction of primary malignant PCs with H/F-LVs was stable. Indeed, GFP expression was maintained in malignant PCs cultured & 2012 Macmillan Publishers Limited

for 1 week together with their bone marrow environment cells in vitro (Figure 4a). Actually, GFP content increased due to continuous synthesis of stable GFP in poorly proliferating primary malignant PCs. HMCLs were also stably transduced with H/F-LVs, as the GFP expression was maintained after 10 -- 14 doubling times, that is, one month of culture (Figure 4b). H/F-LVs also efficiently transduced healthy in-vitro generated plasmablasts or PCs unlike VSV-G-LVs. Respectively, 75% and 39% of healthy plasmablasts and PCs were transduced with H/F-LVs, compared with 19% and 4% for VSV-G-LVs (Pp0.01, Figures 2a -- c). The median mean fluorescence intensity was 10 -- 20-fold higher with H/F-LVs compared to VSV-G-LVs (Pp0.01). As for malignant PCs, H/F-LV transduction did not modify the phenotype of healthy PCs as both GFP þ and GFP PCs expressed the same density of CD138, CD38, CD45 or CD19 (Figure 3b). The transduction of healthy PCs was stable with a maintained GFP expression in PCs cultured for 14 days with stromal cells (Figure 4c). DISCUSSION We show here that measles envelope glycoproteins displaying LVs (H/F-LVs) can efficiently and stably transduce malignant and Leukemia (2012) 1663 - 1670

Plasma cell transduction with measles lentiviruses M Schoenhals et al

1666

4.3%

29.7%

9%

54%

0.5%

59%

Malignant PC transduction

Healthy PC transduction (CD138+) H/F-LV

Percentage of transduced cells

MFI

VSV-G-LV

VSV-G H/F-LV infection

Healthy Plasmablasts

CD138 / DAPI / GFP

Healthy Healthy Healthy PCs Plasmablasts PCs

CD138 / DAPI / GFP

x630 Malignant PC

x630 Healthy PC

Figure 2. Measles H/F lentiviral vectors efficiently transduce plasmablasts, normal and malignant plasma cells. (a) GFP expression in malignant PCs of a representative patient sample, or in healthy PCs or plasmablasts after 3 days transduction with VSV-G-LVs or H/F-LVs (multiplicity of infection, MOI 5) are shown for the CD138 þ cell populations. The percentages of GFP-positive cells are indicated on histograms. Note a shift of the whole histogram in case of H/F-LV transduction likely due to absorption by non-transduced plasma cells of GFP released in the culture medium.34 (b) Percentages and mean fluorescence intensity (MFI)±s.d. of GFP expression in malignant PCs from five patients or healthy PCs and plasmablasts generated from five experiments at day 3 post-transduction with VSV-G-LVs or H/F-LVs (MOI 5). (c) Immunofluorescence staining of malignant or healthy plasma cells transduced with GFP encoding H/F-LVs (MOI 5) and stained with PE-conjugated anti-CD138 mAb and DAPI. Leukemia (2012) 1663 - 1670

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Plasma cell transduction with measles lentiviruses M Schoenhals et al

1667

Plasma cells

GFP

GFP

Primary malignant plasma cells

CD38

CD200

CD19

CD45

GFP

CD138

GFP

Plasma cells

GFP

In vitro generated healthy plasma cells

CD38

CD138

CD45

CD19

Figure 3. The phenotype of primary malignant plasma cells or healthy plasma cells is not modified by measles H/F-lentiviral vector transduction. (a) Malignant PCs of a representative patient sample were transduced with H/F-GFP-LVs (multiplicity of infection, MOI 5) yielding to 21% transduced plasma cells after 3 days of culture. The phenotype of malignant plasma cells was investigated using staining with anti-CD138, CD38, CD200, CD19 or CD45 mAbs. GFP þ transduced malignant plasma cells had the same phenotype as GFP non-transduced ones. Data are from one patient’s sample representative of three. (b) Healthy plasma cells were generated in vitro and transduced at day 7 with H/F-GFP-LVs (MOI 5). After 3 additional days of culture, 16% healthy plasma cells expressed GFP and their phenotype was investigated using staining with anti-CD138, CD38, CD45, CD19 mAbs. GFP þ healthy plasma cells had the same phenotype as GFP non-transduced ones. Data are from one healthy plasma cell sample representative of three. Table 1.

The transduction of human multiple myeloma cell lines with measles envelope pseudotyped-lentivirus did not modify their phenotype LP1 (%)

CD56 CD200 CD27 CXCR4 CD49d CD38 CD138 CD20 CD45 CD19

XG2 (%)

XG20 (%)

H/F-LV-transduced

Non-transduced

H/F-LV-transduced

Non-transduced

H/F-LV-transduced

Non-transduced

64 2 1.9 94.8 100 100 99 0 0.1 0.2

53 1 0.9 90.7 100 100 97 0.3 0.3 0.5

2.4 82 2.5 99.8 100 80 99.6 0.3 1.2 0.6

2.9 65 2 99.8 100 90 99.4 1.15 2 2.3

84.8 99.8 0.2 87 99.9 71.3 99.2 0.3 0.1 0.1

90.3 87.7 2.1 78.7 99.9 81.8 99.7 0.9 1.6 0.9

The LP1, XG2 and XG20 human myeloma cell lines (HMCLs) were transduced with H/F lentivirus and cultured for 1 month. Data show that transduced HMCLs display the same phenotypic characteristics as non-transduced HMCLs.

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Leukemia (2012) 1663 - 1670

Plasma cell transduction with measles lentiviruses M Schoenhals et al

1668 Primary malignant plasma cells cultured for 7 days after H/F-LV transduction Day 2

Day 7 50%

GFP

GFP

43%

CD138

CD138

XG20 cells cultured for 30 days after H/F-LV transduction Day 30

55.7%

GFP

GFP

Day 3

50.7%

SSC

SSC Healthy plasma cells cultured for 14 days after H/F-LV transduction Day 3

Day 14 15.0%

GFP

GFP

16.2%

CD138

CD138

Figure 4. Measles H/F lentivectors stably transduce malignant or healthy plasma cells. (a) Bone marrow mononuclear cells from three patients with MM were transduced with H/F-LVs for 2 days. Cells were washed and malignant PCs were cultured together with their bone marrow environment for 5 days in 200 ml of RPMI1640 culture medium, 10% FCS and IL-6 (2 ng/ml), and the GFP expression was assayed by FACS analysis. Data are those of one patient’s sample, representative of three. Note that GFP content was higher in cultured malignant CD138 þ PCs due to the accumulation of stable GFP in non-proliferating PCs. (b) H/F-LV transduced HMCLs were cultured for 1 month with change of the culture medium every 3 -- 4 days. Cells were harvested every 7 days for assaying the GFP expression by FACS analysis and data are GFP expression at day 3 and day 30 after H/F-LV transduction. (c) The 5  105 in-vitro-generated healthy PCs harvested at day 7 of culture were transduced with H/F-LVs and cultured for 3 days on 104 bone marrow stromal cells in 1 ml of RPMI1640 culture medium, 10% FCS, IL-6 (10 ng/ ml) and IFNa (500 U/ml), washed and cultured for additional 11 days on 104 bone marrow stromal cells in 1 ml of RPMI1640 culture medium, 10% FCS and IL-6 (10 ng/ml). Data are GFP expression in CD138 þ PCs generated in one experiment representative of three at day 3 and day 14 after H/F-LV transduction assayed by FACS analysis. At least 50% PCs died during the additional 11 days of culture, explaining the lower number of viable CD138 þ PCs 14 days after H/F-LV transduction. Note that GFP content was higher in day 14 PCs due to the accumulation of stable GFP in non-proliferating PCs.

healthy PCs, unlike VSV-G pseudotyped LVs. In addition, H/F-LVs did not modify the phenotypic characteristics of healthy or malignant PCs. Both healthy and malignant PCs express the two receptors for measles glycoproteins, SLAMF1/CD150 and CD46, and both receptors were shown to be mandatory to obtain an efficient H/F-LVs transduction in primary lymphocytes.24 Leukemia (2012) 1663 - 1670

The vast majority of primary malignant PCs (495%) is out of the cell cycle26 and their efficient transduction by H/F-LVs confirm recent data showing these H/F-LVs can transduce quiescent human primary B or T lymphocytes.9,10 The poor transduction of healthy PCs by VSV-G-LVs is not related to cell cycle. Indeed, a poor transduction was observed with high cell cycling & 2012 Macmillan Publishers Limited

Plasma cell transduction with measles lentiviruses M Schoenhals et al

plasmablasts as well as with non-cell cycling PCs,19 whereas H/F-LVs efficiently transduce both plasmablasts and PCs. Thus, H/F-LVs provide a powerful tool to stably introduce and express coding DNA in healthy or primary malignant PCs. Many genes whose expression in primary malignant PCs is associated with poor survival are being reported using high throughput microarray methodologies.7,27 - 29 The ability to express these genes in PCs using H/F-LVs will greatly help the understanding of the biological pathways triggered by these gene products and associated with drug resistance and/or poor survival. Another interest will be to confer immunogenicity on primary malignant PCs for the development of immunotherapy strategies, in particular to improve allograft strategies. We and others have shown that malignant PCs of HMCLs are fully efficient to trigger allogenic or autologous cytotoxic T-cell-specific response providing they express immune costimulatory molecules, such as CD80/ B7-1, 4-BB1 ligand or CD40.30 - 32 It is important to extend this finding to primary malignant PCs, introducing these genes using H/F-LVs, in order to promote their use in clinical settings, at least to activate and amplify anti-tumor T cells ex vivo. H/F-LVs should also be promising for the understanding of normal plasma cell biology. Most studies have used murine PCs due to the difficulty to obtain human PCs and to manipulate them.33 The combination of the ability to generate human PCs in vitro and to efficiently induce the expression of a given gene in these cells should make the study of healthy human PCs far easier.19 An interesting application will be to immortalize human PCs to produce human monoclonal antibodies specific to a given antigen, introducing the major genes deregulated in malignant PCs and yielding to HMCLs growing in vitro. In conclusion, the finding that LVs pseudotyped with MV envelope glycoproteins enable an efficient and stable introduction and expression of a given gene in noncycling normal and malignant PCs is a major breakthrough, that will further stimulate the study and understanding of the most important mechanisms controlling normal and malignant PC biology. CONFLICT OF INTEREST The authors declare no conflict of interest.

ACKNOWLEDGEMENTS This work was supported by grants from ARC (SL220110603450, Paris, France), the European Community (FP7- OVERMYR), the ‘Agence Nationale pour la Recherche contre le SIDA et les He´patites Virales’ (ANRS), the ‘Agence Nationale de la Recherche’ (ANR) and the European Community (FP7-HEALTH-2007-B/222878 ‘PERSIST’ and FP7-GENTHALTHER Erare, ERC-2008-AdG-233130-HEPCENT). MS is supported by a grant from the Guillaume Espoir Association (Saint-Genis-Laval, France).

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Supplementary Information accompanies the paper on the Leukemia website (http://www.nature.com/leu)

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