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Induced Expression of B7-1 on Myeloma Cells Following Retroviral Gene Transfer Results in Tumor-Specific Recognition by Cytotoxic T Cells¹

Karin Tarte^*, Xue-Guang Zhang^*,, Eric Legouffe, Catherine Hertog^§, Majid Mehtali^¶, Jean-François Rossi and Bernard Klein^2,*,§

^* Institut National de la Santé et de la Recherche Médicale Unite 475, Montpellier, France; Immunology Research Unit, Suzhou Medical College, Suzhou, China; Service des Maladies du Sang B, Centre Hospitalier Universitaire Montpellier, Hôpital Lapeyronie, Montpellier, France; ^§ Unite de Therapie Cellulaire, Centre Hospitalier Universitaire Montpellier, Hôpital Saint Eloi, Montpellier, France; and ^¶ Transgene, Strasbourg, France

	Abstract

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The aim of this study was to evaluate whether tumor cells from patients with multiple myeloma activate allogeneic and autologous T cells. Results showed that myeloma cells expressed few B7-2 and no B7-1 in six cell lines and primary cells from 11 patients. They expressed substantial levels of HLA class I, CD40, and a set of adhesion molecules. In accordance with the low density of B7 molecules on these cells, they were poor allogeneic CD8⁺ T cell stimulators. Neither IFN- plus TNF- nor CD40 stimulation significantly induced B7-1 or up-regulated B7-2 on human myeloma cell line or primary myeloma cells from six of seven patients. However, such induction was found on autologous bone-marrow nontumoral cells and on autologous dendritic cells following CD40 stimulation. High B7-1 expression was stably obtained on human myeloma cell line using transduction with a B7-1 retrovirus, enabling these cells to stimulate allogeneic CD8⁺, though not CD4⁺, T cell proliferation. For one patient with advanced disease, B7-1 gene transfer made it possible to amplify autologous cytotoxic T cells that killed autologous myeloma cells in an HLA class I-restricted manner, but not autologous PHA blasts. These results suggest that B7-1 gene transfer could be a promising immunotherapeutic approach in multiple myeloma.

	Introduction

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Multiple myeloma (MM)³ is a B cell neoplasia affecting the late stage of B cell differentiation. Tumor plasma clones expand following a multistep transformation process (1) in close contact with the bone-marrow stromal cells expressing adhesion molecules and producing cytokines, especially IL-6, which sustains survival and growth of myeloma cells (2, 3).

Although high-dose chemotherapy and autologous stem cell transplantation have improved the rate of complete remission, some myeloma cells escape treatment and all MM patients relapse (4, 5, 6). The development of immunotherapy approaches designed to get CTL might help to eradicate or control residual tumor cells. The use of professional APC such as dendritic cells (DC) to generate anti-tumoral response is a promising strategy (7, 8). Vaccination of patients with DC pulsed with monoclonal Ig as tumor Ag has already begun in MM (9, 10). DC pulsed with tumor RNA (11, 12, 13) or cell lysates (14) may prove to be useful in obtaining CTL clones against various unidentified Ag. However, this approach is limited in that there is no guarantee that DC will present the same tumor peptides as those presented by the tumor cells.

Alternatively, anti-tumoral CTL can be obtained by using tumor cells themselves as APC. The in vitro or in vivo stimulation of CTL requires TCR activation by a peptide/MHC complex combined with costimulatory signals, in particular CD28 activation by B7 molecules present on APC (15, 16, 17). A high density of B7 molecules is needed to trigger a full T cell response (18, 19). B7-1 (CD80) and B7-2 (CD86) are generally absent or only marginally present on tumor cells but they could be induced upon CD40 ligation or by cytokines such as IFN- in some hemopoietic malignancies (20, 21, 22). In B cell lymphoma and pre-B acute lymphoblastic leukemia, CD40-mediated B7 induction on tumor cells makes it possible to obtain autologous specific anti-tumoral CTL (23, 24). In contrast, in acute myeloid leukemia, CD40 stimulation fails to induce B7-1 and does not increase allogeneic T cell proliferation (20). Myeloma cells express a set of adhesion molecules as well as CD40 and CD28 (25, 26, 27). The myeloma CD28 is able to bind B7 and to activate the phosphatidylinositol 3 kinase (26). Most studies have shown that only a small number of tumor plasma cells express B7-2 and that B7-1 is generally lacking (25, 26, 27, 28, 29, 30, 31, 32). A recent report suggests that the weak B7-1 expression can be doubled after cell culture with IL-6, IFN-, or TNF- (31). CD40 stimulation has been shown to induce IL-6 production and to increase growth and survival of MM cells in vitro (30, 32). In other studies, CD40 stimulation was reported to induce growth arrest (28) and apoptosis (33) on a human myeloma cell line (HMCL). These conflicting results could be explained in part by the use of few cell lines that, sometimes, were not true HMCL but EBV-infected cell lines (ARH 77, HS-Sultan, IM9) (34). Furthermore, research on primary myeloma cells should be developed given the marginal induction of B7 molecules reported and the difficulty of clearly distinguishing myeloma cells from late-stage B cells with anti-CD38 mAb labeling (31). Another way to induce B7 expression is by gene transfer. A recent study reported that the transfer of B7 genes using adeno-associated virus vectors in two HMCL enable them to activate and be lysed by a mixture of allogeneic NK and T cells (35). These effects were due in part to NK cells as previously described for the in vivo rejection of some B7-expressing animal tumors (36, 37).

To date, no study has demonstrated that B7-1⁺ myeloma cells can be used to obtain anti-tumoral autologous T cells in vitro. We show here, using an anti-syndecan-1 mAb that recognizes all viable tumor cells in bone-marrow samples (38, 39, 40), that myeloma cells express no B7-1 and only a few B7-2 molecules. This low B7-2 density was responsible for a weak stimulation of highly purified allogeneic T cells by HMCL. B7-1 was not significantly up-regulated on primary myeloma cells activated by CD40 or IFN- plus TNF- for a majority of patients. In contrast, B7-1 was induced by CD40 ligation on autologous nontumoral bone-marrow cells and on autologous DC. Finally, the transduction of B7-1 on HMCL with a B7-1 retrovirus increased their ability to activate allogeneic CD8⁺ T lymphocytes. For one patient with advanced disease, obtaining B7-1⁺ HMCL made it possible to get specific autologous anti-tumoral CTL.

	Materials and Methods

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Preparation of primary samples

Eleven patients with MM (median age, 66.5 years) were included in this study. There were four IgG, five IgG, one IgA, and one Bence-Jones . Bone-marrow aspirates were collected for the determination of plasma cell labeling index, a marker of disease activity (41), and exceeding cells were used for this study after informed consent. Bone-marrow mononuclear cells (BMMC) were isolated by Ficoll-Hypaque centrifugation (BioWhittaker, Walkersville, MD) and either stained with appropriate mAbs or plated in culture for 4 days. For two patients, purification of syndecan-1⁺ tumor plasma cells was performed with the MI15 anti-syndecan-1 mAb (40) and magnetic cell separation system anti-murine IgG microbeads (Tebu, Le Perray-en-Yvelines, France) as previously described (42). Purified cells contained >92% plasma cells.

Cell lines

Five IL-6-dependent (XG-1, XG-2, XG-6, XG-13, and XG-14) and one autonomous (RPMI 8226) HMCL were used in this study. These cell lines were true myeloma cell lines with a plasma cell phenotype, no expression of B cell markers, and no infection with EBV. XG-1, XG-2, XG-6, and XG-13 had been previously established in our laboratory (29, 43), and RPMI 8226 was purchased from American Type Culture Collection (Manassas, VA). They were cultured in RPMI 1640 supplemented with 10% FCS, 2 mM L-glutamine, and 5 x 10^-5 M 2-ME, and with 3 ng/ml of human IL-6 for XG-1, XG-2, XG-6, and XG-13. XG-14 was cultured in X-VIVO 20 serum-free medium (BioWhittaker) supplemented with 3 ng/ml of human IL-6. L cells stably transfected with CD40 ligand (CD40L) were kindly provided by Sem Saeland (Schering-Plough, Dardilly, France).

Phenotypic analysis

Expression of cell surface molecules on HMCL and B7-1 HMCL was determined using FITC-conjugated anti-CD28, anti-CD54, anti-CD58, anti-CD11a, anti-CD18, anti-HLA-ABC, anti-HLA-DR (Immunotech, Marseille, France), and anti-B7-2 (PharMingen, San Diego, CA) and PE-conjugated anti-B7-1, anti-Fas, and anti-CD40 mAbs (Immunotech). For primary BMMC, double staining of malignant plasma cells was performed using the same mAbs and FITC- or biotin-conjugated anti-syndecan-1 mAb (MI15). All fluorescence analyses were conducted on a FACScan apparatus (Becton Dickinson, San Jose, CA). Negative controls were done with corresponding irrelevant isotype-matched murine mAbs.

Induction of costimulatory molecules on myeloma cells

HMCL were cultured at 2 x 10⁵/ml and BMMC and purified syndecan-1⁺ plasma cells at 1 x 10⁶/ml in RPMI 1640 supplemented with 10% FCS, 2 mM L-glutamine, and 5 x 10^-5 M 2-ME, except for the XG-14 cell line, which was cultured in X-VIVO 20 medium. All cultures were performed in the presence of 3 ng/ml of human IL-6. Cells were stimulated with either IFN- (100 U/ml; Genzyme, Cambridge, MA) plus TNF- (20 ng/ml; R&D Systems, Minneapolis, MN) or cocultured with CD40L-transfected L cells (1 x 10⁵/ml). As a control of the specificity of CD40 stimulation, we used nontransfected L cells (1 x 10⁵/ml). After 4 days of culture in these different conditions (IL-6, IL-6 plus IFN- plus TNF-, IL-6 plus L cells, IL-6 plus CD40L-transfectant), cells were harvested and simple (HMCL and purified primary plasma cells) or double (BMMC) stainings were done as described above.

Obtention of HMCL transfected with B7-1 cDNA (B7-1 HMCL)

The full-length human B7-1 cDNA was subcloned into the neomycin-selectable 5192 retroviral vector with EcoRI/XhoI cloning sites and transfected into the E293 packaging cell line using Lipofectamine (Life Technologies, Paisley, U.K.). The packaging cell line was also transfected with the empty retroviral vector. The 5192 retroviral vector and the E293 packaging cell line were a gift from Dr. Mehtali (Transgene, Strasbourg, France). Transfected cells were selected by outgrowth with 600 µg/ml of G418 (Life Technologies) for 10 days. Resistant cells expressing the B7-1 molecule were sorted using a Vantage cell sorter (Becton Dickinson), cultured at a high cell density, and the supernatant was filtered and stored at -70°C. Culture supernatant of G418 resistant clones of E293 packaging cells transfected with the empty vector was also collected, filtered, stored at -70°C, and used as a source of control retrovirus. HMCL were plated at 10⁶ cells/well in six-well dishes and exposed to control or B7-1 retroviral supernatant for 2 h in the presence of 8 µg/ml of polybrene (Sigma, St. Louis, MO). After two cycles of infection, cells were selected with 1200 µg/ml of G418 over a period of 10 days. Resistant clones obtained with the B7-1 retrovirus were analyzed for surface expression of B7-1 and sorted as described above. The stable transfected HMCL obtained with the B7-1 retrovirus (named B7-1 XG-1, B7-1 XG-6, and B7-1 XG-14) and with the control retrovirus (named Neo XG-1, Neo XG-6 and Neo XG-14) were subsequently maintained with 500 µg/ml of G418.

Allogeneic MLR (allo-MLR)

Heparinized venous peripheral blood from healthy volunteers was collected after written informed consent. T lymphocytes were purified by depletion of non-T cells with mAbs (Immunotech) and magnetic beads (Dynal, Oslo, Norway) as described (44). Briefly, monocytes and B cells were first depleted using CD14- and CD19-coated beads. Then, non-T cells were removed by incubating with a mixture of CD16, CD56, and HLA-DR mAbs and then goat anti-mouse Ig beads. After two rounds of purification, the purity of resting CD3⁺ cells was always >98%. CD4⁺ and CD8⁺ T cells were further purified by removing CD8⁺ or CD4⁺ cells using anti-CD8 and anti-CD4 mAbs and magnetic beads. The purity of the T cell subpopulations was >98%. Cells were frozen in 50% FCS-10% DMSO at 20 x 10⁶ cells per vial. To evaluate HMCL-induced T cell proliferation, primary allo-MLR was performed using mitomycin C (Sigma)-treated (50 µg/ml) HMCL or B7-1 HMCL cocultured at 5 x 10⁴ cells/well with 10⁵ allogeneic T cells in 96-well round-bottom plates in RPMI 1640 and 5% heat-inactivated human AB serum. When indicated, antagonist (CD28.6) anti-CD28 (provided by Daniel Olive, Marseille, France), anti-HLA-ABC, or anti-B7-2 mAbs or their isotypic controls were added at 10 µg/ml. After 5 days of coculture, cells were pulsed with 1 µCi of [³H]TdR (sp. act. 25 Ci/ml; Amersham, Buckingham, U.K.) for 12 h, harvested, and counted. All microculture tests were conducted in triplicate and stimulation indexes (SI) were calculated as follows: SI = cpm_{(T cells + HMCL)}/cpm_{(T cells)} + cpm_(HMCL). To quantify T cell amplification and activation, bulk cocultures of mitomycin-treated HMCL, neo HMCL, or B7-1 HMCL and purified T cells were performed at a responder:stimulator cell ratio of 3:1. Cells were seeded at 1.5 x 10⁶ cells/ml in RPMI 1640 and 5% AB serum in 6-well plates. At days 2 and 5 of culture, cells were counted and used for phenotypic analysis with FITC-anti-CD25 and FITC-anti-HLA-DR mAbs (Immunotech). At the same time, culture supernatants were collected and stored at -20°C for IL-2 measurement using a commercially available ELISA test (Genzyme).

Generation of DC

For two patients, DC were generated from adherent apheresis cells as described (44). Briefly, apheresis cells were collected after mobilization of hemopoietic precursors with high dose cyclophosphamide (4 g/m²) and recombinant human G-CSF (rhG-CSF, filgrastim, Neupogen; Amgen-Roche, Neuilly-sur-Seine, France; daily injection of 5 µg/kg) and plated for 2 h at 37°C. Nonadherent cells were discarded and adherent cells were then cultured for 7 days in X-VIVO 15 medium (BioWhittaker) with 2% human albumin, 1000 U/ml of rhGM-CSF (Leucomax; Sandoz, Basel, Switzerland) and 500 U/ml of rhIL-4 (Genzyme). Cells harvested at day 7 exhibited the typical morphological features of DC and expressed HLA class I, HLA class II, B7-1, B7-2, CD4, CD40, and no CD14 as reported (44). They were cultured at 4 x 10⁵/ml in the presence of 4 x 10⁴ nontransfected L cells or CD40L-transfected L cells/ml. After 3 days of culture, cells were stained with FITC-anti-B7-1 or control murine mAbs.

Generation of autologous anti-tumoral T cells

For one patient with plasma cell leukemia (PCL), PBMC (comprising 30% CD3⁺ T cells, 40% syndecan-1⁺ tumor cells, and 15% CD16⁺ NK cells) were frozen at diagnosis and an autologous HMCL was established (XG-14) and transduced with control vector (Neo XG-14) and B7-1 retrovirus (B7-1 XG-14). PBMC (1.5 x 10⁶/ml) were stimulated with mitomycin-treated (50 µg/ml) HMCL, Neo HMCL, or B7-1 HMCL at an E:T ratio of 2:1 in RPMI 1640 supplemented with 5% human AB serum for 6 days. Dead cells were eliminated by Ficoll-Hypaque centrifugation. Viable cells were then cultured at 7.5 x 10⁵/ml and restimulated on days 6, 12, 18, and 24 with mitomycin-treated HMCL, Neo HMCL, or B7-1 HMCL at an E:T ratio of 2:1 in the presence of IL-2 (30 U/ml) and IL-12 (1 ng/ml) (R&D Systems). Cytokines were added on days 7, 13, 19, and 25 of culture. When indicated, IL-7 was added at 5 ng/ml. Cell-mediated toxicity was determined on day 28 using a standard 4-h ⁵¹Cr release (Amersham) assay using E:T ratios of 3:1, 10:1, and 30:1. XG-14 (autologous HMCL), XG-2 (allogeneic HMCL), PHA-blasts (autologous nontumoral cells), and K562 were used as target cells at 5000 cells/well in 96-well plates. All experiments were performed in triplicate. To determine whether the cytolysis was restricted by HLA class I, target cells were preincubated with 10 µg/ml of anti-HLA class I mAb. After a 4-h incubation at 37°C, supernatants were harvested and radioactivity was measured with a counter. The percentage of specific lysis was determined as: (experimental ⁵¹Cr release - spontaneous ⁵¹Cr release)/(maximum ⁵¹Cr release - spontaneous ⁵¹Cr release) x 100. The ratio (spontaneous release/maximum release) was always below 30%.

	Results

Top Abstract Introduction Materials and Methods Results Discussion References

 
Expression of adhesion and costimulatory molecules on HMCL and primary myeloma cells

Because the expression of adhesion and costimulatory molecules on APC plays a major role in the activation of T cells, we first investigated it on six HMCL (Table I) and on bone-marrow plasma cells from 11 MM patients. To label patient tumor cells, we used an anti-syndecan-1 mAb that stains all viable myeloma cells in tumor samples (38, 39, 40). The phenotype obtained with patient 4’s cells is outlined on Fig. 1, and data obtained for the 11 patients are summarized in Table II. Concerning adhesion molecules, CD54 was expressed at a high level and CD58 at an intermediate level by all HMCL (percentage of positive cells, 100%; mean fluorescence intensity (MFI), 158–710 for CD54 and 21–54 for CD58) and primary tumor samples (mean percentage of positive cells, 83.3 ± 19.9% for CD54 and 47 ± 30.8% for CD58). CD11a and CD18 were expressed on 2 of 6 HMCL, on <20% of tumor cells in 6 of 11 fresh samples, and were virtually absent in other samples. HLA class I was largely present on all plasma cells. HLA-DR was absent on HMCL and marginally present on patient tumor cells (mean percentage of positive cells, 9.4 ± 8.2%). In addition, in keeping with results obtained with HMCL, primary tumor cells expressed no detectable B7-1 and a variable level of B7-2 (mean percentage of positive cells, 38.6 ± 37.1%). It should be noted, as previously reported (25, 27), that the CD28 molecule was present on all cell lines and on 10 of 11 patient tumor samples. CD40 expression was heterogeneous in HMCL but was always present on tumor samples (mean percentage of positive cells, 93.3 ± 8.4%; mean MFI, 155 ± 90). Thus the phenotype of HMCL and patients’ myeloma cells was very similar. In particular, myeloma cells expressed CD54 and CD58, HLA class I, CD40, no B7-1, and few B7-2.

View larger version (48K): [in this window] [in a new window]   FIGURE 1. Expression of adhesion and costimulatory molecules on primary myeloma cells. BMMC from patient 4 were double-stained either with biotin-MI15 plus PE-streptavidin (FL-2) and FITC-conjugated anti-CD54, CD58, CD11a, CD18, HLA-ABC, HLA-DR, B7-2, CD28 mAbs or with FITC-MI15 (FL-1) and PE-conjugated anti-B7-1, CD40 mAbs. Irrelevant mouse Ig were used as negative controls as shown on the last two plots. On each dot plot, the percentages of syndecan-1+ and syndecan-1- cells expressing the cell-surface markers were indicated in the upper right and lower right panels, respectively.

When XG-1, XG-6, and XG-14 were used as stimulator cells in primary allo-MLR, a small but significant T cell proliferation was induced with a mean SI of 9 for XG-1 (range, 3–20; n = 7), 5 for XG-6 (range, 4–8; n = 4), and 8 for XG-14 (range, 7–11; n = 3), depending on the various donors of allogeneic T cells (not shown). This activation was inhibited by an anti-HLA-ABC mAb, indicating that it was mediated through allopeptide presentation by MHC class I molecules. In addition, it was also inhibited by antagonist anti-CD28 or anti-B7-2 mAbs, suggesting that it was mediated in part through the activation of T cell CD28 by the myeloma B7-2 (Fig. 2).

View larger version (20K): [in this window] [in a new window]   FIGURE 2. Stimulation of allogeneic CD3+, CD8+, and CD4+ T cell proliferation in response to XG-1 and B7-1 XG-1. A total of 5 x 104 mitomycin-treated XG-1 and B7-1 XG-1 were used as stimulator cells for 105 highly purified allogeneic CD3+, CD8+, or CD4+ T cells. When indicated, 10 µg/ml of anti-HLA-ABC, antagonist anti-CD28 (anti-CD28N), or anti B7-2 mAbs were added. Cell proliferation was evaluated by a 12-h pulse with [3H]TdR after 5-day cocultures. No inhibition was found with control mouse mAbs. Data are the mean ± SD of the [3H]TdR incorporations determined on sextuplate culture wells. In most groups, [3H]TdR incorporation and SD were too small to be visible on the graph.

Stimulation of CD40 is known to induce B7-1 expression on normal B cells and DC as well as in several tumor cells from patients with B cell lymphoma or B cell leukemia (21, 22). IFN- and TNF- may also increase costimulatory molecule expression (20, 31). To enhance the Ag-presenting capacity of myeloma cells, we looked for the effect of IFN- plus TNF- and CD40 stimulation on the expression of costimulatory molecules. As outlined in Table III, the association of IFN- and TNF- induced a low level of B7-1 and a high level of HLA-DR on the RPMI 8226 cell line. However, these molecules remained undetectable on the other cell lines stimulated by these cytokines. As a control, Fas was up-regulated on HMCL upon IFN- plus TNF- stimulation, and this was associated with an increased sensitivity to Fas-mediated apoptosis for XG-1 and XG-2 cell lines (Table III and data not shown). The density of CD54 was also significantly up-regulated on four of the five HMCL tested, as can be seen in Table III. B7-2, CD11a, CD18, and CD58 were not modulated by IFN- plus TNF- stimulation (data not shown). Independent of their level of CD40 expression, it was impossible to induce B7-1, HLA-DR, or adhesion molecules on HMCL by coculture with CD40L-transfected cells (Table III and data not shown).

View this table: [in this window] [in a new window]   Table III. Induction of B7-1, HLA-DR, Fas, and CD54 on HMCL by IFN- and TNF- or by the CD40L transfectant1

View larger version (31K): [in this window] [in a new window]   FIGURE 3. Neither IFN- plus TNF- nor CD40 stimulation could induce B7-1 on nonpurified primary myeloma cells. BMMC were cultured at 106/ml in RPMI 1640–10% FCS supplemented with IL-6 (medium) and were stimulated with IFN- plus TNF- or CD40L-transfected L cells. Results obtained with nontransfected L cells were identical with those obtained in medium alone and are not shown. After 4 days, cells were harvested and double-stained as described in Fig. 1. The percentages of B7-1+ syndecan-1+ and B7-1+ syndecan-1- cells are indicated in the upper right and the lower right quadrant, respectively.

View larger version (25K): [in this window] [in a new window]   FIGURE 4. Neither IFN- plus TNF- nor CD40 stimulation induced B7-1 on purified primary myeloma cells, unlike autologous DC. A, Syndecan-1+ purified myeloma cells (patient 5) were cultured at 106/ml in medium alone or in the presence of IFN- plus TNF-, nontransfected L cells, or CD40L-transfected L cells. B, Autologous DC (patient 5) obtained after 7 days of culture of adherent apheresis cells in X-VIVO 15 medium supplemented with 2% human albumin, GM-CSF, and IL-4, were cultured at 4 x 105/ml with nontransfected or CD40L-transfected L cells. Myeloma cells and DC were harvested after 3–4 days of stimulation and were stained with anti-B7-1 mAbs (bold lines) or isotype-matched control murine mAbs (dotted lines). The same data were obtained with patient 2’s cells.

After transduction of XG-1, XG-6, and XG-14 HMCL with the B7-1 retrovirus, selection, and sorting, we obtained B7-1 HMCL that expressed high levels of the B7-1 molecule without any modification in the pattern of expression of the other costimulatory or adhesion molecules (CD54, CD58, CD11a, CD18, B7-2, CD40; data not shown). Neo HMCL transduced with an empty control retrovirus did not express B7-1 and had a similar phenotype as that described in Table I for parental cells (data not shown). B7-1 HMCL were four to five times more potent for stimulating allogeneic CD3⁺ T cells than parental HMCL with a mean SI of 41 for B7-1 XG-1 (range, 10–99; n = 7); 26 for B7-1 XG-6 (range, 10–31; n = 5), and 13 for B7-1 XG-14 (range, 9–20; n = 4) (data not shown). As detailed in Fig. 2, B7-1 XG-1 was unable to activate CD4⁺ T cells, in agreement with the absence of HLA class II expression, but it induced a high level of proliferation of purified CD8⁺ T cells, a proliferation inhibited by an anti-HLA-ABC mAb. The level of CD8⁺ T cell proliferation induced by B7-1 XG-1 was not different from that induced by PHA. In addition, B7-1 XG-1 induced purified CD8⁺ T cells but not purified CD4⁺ T cells to express HLA-DR and CD25 (data not shown) and to produce IL-2 (Table V). In contrast, parental XG-1 and Neo XG-1 induced only a weak expression of CD25 and HLA-DR on CD8⁺ T cells with no measurable IL-2 production (data not shown and Table V). Similar data were obtained with XG-6, Neo XG-6, and B7-1 XG-6 as well as XG-14, Neo XG-14, and B7-1 XG-14 HMCL (data not shown).

For one patient with a PCL at diagnosis, PBMC containing 30% CD3⁺ T cells (15% CD4⁺ and 15% CD8⁺ T cells) and 40% syndecan-1⁺ myeloma cells were harvested and cultured with IL-6. An IL-6-dependent autologous HMCL (XG-14) was obtained and transduced with the B7-1 retrovirus (B7-1 XG-14) and with the empty retrovirus (Neo XG-14). XG-14 cells expressed the same adhesion and costimulatory molecules as the primary patient myeloma cells (Table I and data not shown). As mentioned in Table I, neither IFN- plus TNF- nor CD40 stimulation induced B7-1 on XG-14 HMCL but transduction with B7-1 retrovirus induced high B7-1 expression and strong alloreactivity. We then tested to see whether the transduction of the B7-1 molecule made it possible to generate specific anti-tumoral T cells. Twenty million nonpurified PBMC (containing 6 million CD3⁺ T cells) were cocultured with 10 million irradiated XG-14, Neo XG-14, and B7-1 XG-14. Cells were restimulated on days 6, 12, 18, and 24 with XG-14, Neo XG-14, or B7-1 XG-14. IL-2 and IL-12 were added only after the second stimulation on days 7, 13, 19, and 25. Neither parental XG-14 nor Neo XG-14 supported the growth of autologous T lymphocytes because all the cells died before day 12 (Fig. 5). On the contrary, B7-1 XG-14 allowed a great amplification of CD3⁺ CD8⁺ TCR ß⁺ CD16^- cells (Fig. 6). After 28 days of culture, CD8⁺ T cells had expanded by 15-fold. When these cells were used in a standard ⁵¹Cr release assay, they efficiently killed autologous HMCL (XG-14) but not the NK-sensitive K562 cell line nor autologous PHA blasts. The allogeneic XG-2 cell line was only marginally killed, and the lysis of XG-14 was blocked by anti-HLA-ABC mAb (Fig. 7), indicating that we have obtained HLA-restricted anti-tumoral cytotoxic T cells for this patient suffering from a very aggressive disease. After 35 days of culture, CD8⁺ T cells could not be further expanded and they progressively died within 15 days. Of note, in our culture conditions, i.e., nonpurified T cells with a great tumoral (40% syndecan-1⁺) and NK (15% CD16⁺) cell contamination, the addition of cytokines at the beginning of the culture or the use of IL-7 led to a proliferation of CD16⁺ NK cells and TCR ⁺ T cells (data not shown).

View larger version (11K): [in this window] [in a new window]   FIGURE 5. B7-1 gene transfer allows the expansion of autologous T cells. PBMC (1.5 x 106/ml) were cultured at an E:T ratio of 2:1 with autologous mitomycin-treated HMCL (XG-14), Neo HMCL (Neo XG-14), or B7-1 HMCL (B7-1 XG-14) in RPMI 1640–5% AB serum. Every 6 days, cells were harvested, counted, and restimulated with the appropriate cell line. IL-2 and IL-12 were added after the second boost.

View larger version (27K): [in this window] [in a new window]   FIGURE 6. Phenotype of autologous T cell line. After 28 days of coculture with B7-1 XG-14 (five stimulations), T cells were stained with anti-CD3, -CD4, -CD8, -CD16, -TCRß, and -TCR mAbs (bold lines). Controls (dotted lines) were isotype-matched irrelevant Abs.

View larger version (14K): [in this window] [in a new window]   FIGURE 7. Cytotoxicity of CTL expanded by stimulation with autologous B7-1 HMCL. Cytotoxicity was measured at day 28 using a standard 4-h 51Cr release assay. Autologous XG-14 HMCL, allogeneic XG-2 HMCL, and autologous nontumoral PHA-blasts were used as target. When indicated, cells were preincubated with anti-HLA-ABC mAb at 10 µg/ml.

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion References

We first analyzed 6 HMCL and 11 bone-marrow tumoral samples for their expression of adhesion and costimulatory molecules. As previously described (25, 26, 27, 28, 29, 30, 31, 32), myeloma cells expressed CD28, HLA class I, CD54, CD58, and CD40, and some of them expressed low levels of CD11a and CD18. In addition, primary plasma cells expressed few HLA-DR molecules. With regards to B7 molecules, we found a variable expression of B7-2 (38.6 ± 37.1%) but no detectable B7-1. However, Yi et al. recently reported that 3.5% of patient myeloma cells expressed B7-1 (31). This study was conducted using CD38 Ag as a tumor marker. CD38 is highly expressed on primary myeloma cells (47, 48, 49) but it is also expressed on a large proportion of normal BMMC (50). Thus, it cannot be excluded that a minority of the bone-marrow nontumoral cells might express a high level of CD38, especially after 3 days of culture with various activation signals, and therefore account for the very weak B7-1 positivity found by Yi et al. on myeloma cells (<5%). To study the phenotype of primary myeloma cells, we used an anti-syndecan-1 mAb. Indeed, we and others have shown that syndecan-1 is present only on viable myeloma cells, unlike nontumor cells (38, 39, 40). We have recently confirmed that all syndecan-1⁺ cells had a plasma-cell morphology using serial histological sections of decalcified bone of MM patients (51). However, as with the study conducted by Yi et al. mentioned above, we could not exclude that a minority of syndecan-1⁺ cells were not myeloma cells; consequently, results below 5% must be considered cautiously. The low-level expression of B7-2 and the lack of B7-1 may explain the poor ability of HMCL and purified primary myeloma cells to induce CD8⁺ allogeneic T cell activation, IL-2 production, and proliferation. Similar results were reported for pre-B acute lymphoblastic leukemia cells (21) and acute myeloid leukemia cells (20) and are in line with the demonstration that B7 density is critical to promote an effective T cell response, especially for CD8⁺ T lymphocytes (18, 19).

To up-regulate B7 expression on myeloma cells, we tested two strategies of activation: by IFN- plus TNF- and by CD40 ligand. Indeed, IFN- and TNF- increase B7-1 expression on normal cells as well as on acute myeloid leukemia cells (20). In addition, Yi et al. reported that B7-1 was induced on 14.3% of patient CD38⁺⁺ bone-marrow cells by IL-6 and on 13.3% by IFN- (31). In the present study’s culture conditions, i.e., in the presence of IL-6, no B7-1 expression was induced by IFN- plus TNF- stimulation on HMCL, except for the RPMI 8226 cell line on which HLA-DR was induced as well. This result suggests that B7-1 is induced by cytokines in only rare cases. We found that IFN- induced apoptosis of primary myeloma cells as previously reported (45, 46), despite the presence of IL-6 and TNF-, both survival factors for myeloma cells (2, 52). In addition, no B7-1 induction nor B7-2 up-regulation was observed after culture with IFN- plus TNF- on the remaining syndecan-1⁺ viable cells. Thus, the use of IFN- plus TNF- is not an effective strategy for inducing high levels of B7 molecules on the majority of myeloma cells. Of further interest, Fas and CD54 were induced by IFN- plus TNF- on some HMCL and primary tumor samples, as recently reported (53).

We then used CD40 stimulation, known to increase B7-1 expression on several B cell neoplasia (21, 22) as well as on normal B cells (54) and DC (55). As with data reported for the XG-2 cell line (28), we showed that B7 molecules were not significantly induced by CD40 stimulation on myeloma cells from five of five cell lines and primary myeloma cells from six of seven patients. Investigation is needed into whether additional costimulatory signals (provided by cytokines or activation molecules) could overlap the defect of B7-1 induction by CD40 activation. For only one patient, 12% of myeloma cells expressed B7-1 and HLA-DR following CD40 stimulation. In contrast, B7-1 was induced by CD40 stimulation on syndecan-1^- nontumoral cells cultured with tumor cells. For two patients, CD40 stimulation also up-regulated B7-1 on DC, whereas it had no effect on purified myeloma cells. No adhesion nor costimulatory molecule could be induced on HMCL by CD40 stimulation. However, CD54 and Fas molecules were up-regulated by CD40 ligation on most primary plasma cells. It is not possible to conclude presently whether this induction of Fas and CD54 is due to a direct activation of myeloma CD40 or to an indirect activation of CD40⁺ nonmyeloma cells and a secondary secretion of inflammatory mediators, especially TNF- (56, 57).

Transferring the B7-1 gene is an alternative way to obtain myeloma cells expressing a high density of the B7-1 molecule. By using a B7-1 retrovirus, myeloma cell lines with a stable and high expression of B7-1 were obtained. B7-1 myeloma cells efficiently activated allogeneic CD8⁺ cells but failed to activate CD4⁺ T cells because they lacked expression of HLA class II molecules. T cell activation was evidenced by an early induction of activation markers (CD25 and HLA-DR), production of IL-2, and proliferation that was inhibited by anti-CD28 mAb. The production of IL-2 was transient, as previously reported for the activation of CD8⁺ T cells (18). In a recent study, Wendtner et al. showed that the transfer of B7 genes in two HMCL using adeno-associated viral vectors made it possible to activate allogeneic PBMC containing nonpurified preactivated NK and T cells (35). The authors state that this effect was due not only to a specific activation of TCR through allo-peptide presentation by MHC molecules on myeloma cells but also to the activation of allogeneic NK cells. Contrary to this study, we used highly purified resting T cells and showed that their activation was mediated through HLA class I/TCR interaction.

Of major interest in getting B7-1 myeloma cells is the generation of anti-tumoral autologous T cells. For one patient, we were able to get T cells at diagnosis and a myeloma cell line (XG-14) at the terminal stage of the disease. XG-14 could be then transduced with the B7-1 retrovirus (B7-1 XG-14). CD8⁺ TCRß⁺ cells could be expanded by coculture with B7-1 XG-14 in the presence of both IL-2 and IL-12 added after the second stimulation. No T cell amplification occurred with parental XG-14 or Neo XG-14 as stimulator cells. The resulting T cells were able to lyse XG-14 but neither allogeneic HMCL nor autologous PHA T cell blasts. However, these data need to be confirmed with other patient primary cells before concluding that primary myeloma cells transduced with B7-1 are efficient autologous APC. Such experiments are seriously limited by the difficulty in obtaining large numbers of primary myeloma cells. Indeed, myeloma cell lines can only be obtained from patients with terminal plasma cell leukemia. This difficulty is further compounded by the multiple chemotherapy regimen and corticoid treatments that most likely delete anti-tumoral T cells. For patients with chronic disease, we have developed a method to purify large numbers of syndecan-1⁺ plasma cells from bone-marrow using B-B4 or MI15 anti-syndecan-1 mAbs (42). In addition, several studies have reported the efficiency of adenovirus-mediated gene transfer in primary myeloma cells (58, 59). If the early harvesting of large bone-marrow samples soon after diagnosis for purifying myeloma cells is deemed as ethical, the development of T cell activation by modified primary myeloma cells will be possible.

Another strategy would be to use DC, which are phenotypically and functionally identical both in MM patients and in healthy individuals (44, 60), and to pulse them with identified tumor Ags (9, 10, 61), cell lysates, or tumor RNA. Even if DC are undoubtedly the best APC, there is no guarantee that they will present the same tumor peptides through MHC class I as those presented by tumor cells. Thus, it is not yet possible to predict which of these two strategies will be the most promising for generating specific anti-tumor CTL in MM patients.

	Footnotes

 
¹ This work was supported by grants from Ligue Nationale Contre le Cancer, Axe Immunologie des tumeurs, Paris, France.

² Address correspondence and reprint requests to Dr. Bernard Klein, Institut National de la Santé et de la Recherche Médicale Unite 475, 99 rue Puech Villa, 34197 Montpellier Cedex 5, France. E-mail address:

³ Abbreviations used in this paper: MM, multiple myeloma; BMMC, bone-marrow mononuclear cells; DC, dendritic cells; HMCL, human myeloma cell line; MFI, mean fluorescence intensity; PCL, plasma cell leukemia; SI, stimulation index. CD40L, CD40 ligand; allo-MLR, allogeneic MLR; rh, recombinant human.

Received for publication October 29, 1998. Accepted for publication April 14, 1999.

	References

Top Abstract Introduction Materials and Methods Results Discussion References

 

1. Hallek, M., P. Leif Bergsagel, K. C. Anderson. 1998. Multiple myeloma: increasing evidence for a multistep transformation process. Blood 91:3.[Free Full Text]
2. Klein, B., X. G. Zhang, Z. Y. Lu, R. Bataille. 1995. Interleukin-6 in human multiple myeloma. Blood 85:863.[Free Full Text]
3. Lichtenstein, A., Y. P. Tu, C. Fady, R. Vescio, J. Berenson. 1995. Interleukin-6 inhibits apoptosis of malignant plasma cells. Cell Immunol. 162:248.[Medline]
4. Attal, M., J. L. Harousseau, A. M. Stoppa, J. J. Sotto, J. G. Fuzibet, J. F. Rossi, P. Casassus, H. Maisonneuve, T. Facon, N. Ifrah, C. Payen, R. Bataille. 1996. A prospective randomized trial of autologous bone marrow transplantation and chemotherapy in multiple myeloma. N. Engl. J. Med. 335:91.[Abstract/Free Full Text]
5. Harousseau, J. L., M. Attal, M. Divine, G. Marit, V. Leblond, A. M. Stoppa, J. H. Bourhis, D. Caillot, M. Boasson, J. F. Abgrall, T. Facon, C. Linassier, J. Y. Cahn, T. Lamy, X. Troussard, N. Gratecos, B. Pignon, G. Auzanneau, R. Bataille. 1995. Autologous stem cell transplantation after first remission induction treatment in multiple myeloma: a report of the french registry on autologous transplantation in multiple myeloma. Blood 85:3077.[Abstract/Free Full Text]
6. Bjorkstrand, B., P. Ljungman, J. M. Bird, D. Samson, G. Gahrton. 1995. Double high-dose chemoradiotherapy with autologous stem cell transplantation can induce molecular remissions in multiple myeloma. Bone Marrow Transplant. 15:367.[Medline]
7. Schuler, G., R. M. Steinman. 1997. Dendritic cells as adjuvants for immune-mediated resistance to tumors. J. Exp. Med. 186:1183.[Free Full Text]
8. Hart, D. N. J.. 1998. Dendritic cells: unique leukocyte populations which control the primary immune response. Blood 90:3245.[Free Full Text]
9. Reichardt, V., C. Okada, C. Benike, G. Long, E. Engleman, K. Blume, R. Levy. 1996. Idiotypic vaccination using dendritic cells for multiple myeloma patients after autologous peripheral blood stem cell transplantation. Blood 88:481a. (Abstr.).
10. Wen, Y. J., M. Ling, R. Bailey-Wood, S. H. Lim. 1998. Idiotypic protein-pulsed adherent peripheral blood mononuclear cell-derived dendritic cells prime immune system in multiple myeloma. Clin. Cancer Res. 4:957.[Abstract]
11. Boczkowski, D., S. K. Nair, D. Snyder, E. Gilboa. 1996. Dendritic cells pulsed with RNA are potent antigen-presenting cells in vitro and in vivo. J. Exp. Med. 184:465.[Abstract/Free Full Text]
12. Ashley, D. M., B. Faiola, S. Nair, L. P. Hale, D. D. Bigner, E. Gilboa. 1997. Bone marrow-generated dendritic cells pulsed with tumor extracts or tumor RNA induce antitumor immunity against central nervous system tumors. J. Exp. Med. 186:1177.[Abstract/Free Full Text]
13. Nair, S. K., D. Boczkowski, M. Morse, R. I. Cumming, H. K. Lyerly, E. Gilboa. 1998. Induction of primary carcinoembryonic antigen (CEA)-specific cytotoxic T lymphocytes in vitro using human dendritic cells transfected with RNA. Nat. Biotechnol. 16:364.[Medline]
14. Nestle, F. O., S. Alijagic, M. Gilliet, Y. Sun, S. Grabbe, R. Dummer, G. Burg, D. Schadendorf. 1998. Vaccination of melanoma patients with peptide- or tumor lysate-pulsed dendritic cells. Nat. Med. 4:328.[Medline]
15. Linsley, P. S., W. Brady, L. Grosmaire, A. Aruffo, N. K. Damle, J. A. Ledbetter. 1991. Binding of the B cell activation antigen B7 to CD28 costimulates T cell proliferation and interleukin 2 mRNA accumulation. J. Exp. Med. 173:721.[Abstract/Free Full Text]
16. Gimmi, C. D., G. J. Freeman, J. G. Gribben, K. Sugita, A. S. Freedman, C. Morimoto, L. M. Nadler. 1991. B-cell surface antigen B7 provides a costimulatory signal that induces T cells to proliferate and secrete interleukin 2. Proc. Natl. Acad. Sci. USA 88:6575.[Abstract/Free Full Text]
17. Harding, F. A., J. P. Allison. 1993. CD28–B7 interactions allow the induction of CD8⁺ cytotoxic T lymphocytes in the absence of exogenous help. J. Exp. Med. 177:1791.[Abstract/Free Full Text]
18. Deeths, M. J., M. F. Mescher. 1997. B7-1-dependent co-stimulation results in qualitatively and quantitatively different responses by CD4⁺ and CD8⁺ T cells. Eur. J. Immunol. 27:598.[Medline]
19. Dorfman, D. M., J. L. Schultze, A. Shahsafaei, S. Michalak, J. G. Gribben, G. J. Freeman, G. S. Pinkus, L. M. Nadler. 1997. In vivo expression of B7-1 and B7-2 by follicular lymphoma cells can prevent induction of T-cell anergy but is insufficient to induce significant T-cell proliferation. Blood 90:4297.[Abstract/Free Full Text]
20. Costello, R. T., F. Mallet, D. Sainty, D. Maraninchi, J. A. Gastaut, D. Olive. 1998. Regulation of CD80/B7-1 and CD86/B7-2 molecule expression in human primary acute myeloid leukemia and their role in allogenic immune recognition. Eur. J. Immunol. 28:90.[Medline]
21. Cardoso, A. A., J. L. Schultze, V. A. Boussiotis, G. J. Freeman, M. J. Seamon, S. Laszlo, A. Billet, S. E. Sallan, J. G. Gribben, L. M. Nadler. 1996. Pre-B acute lymphoblastic leukemia cells may induce T-cell anergy to alloantigen. Blood. 88:41.[Abstract/Free Full Text]
22. Schultze, J. L., A. A. Cardoso, G. J. Freeman, M. J. Seamon, J. Daley, G. S. Pinkus, J. G. Gribben, L. M. Nadler. 1995. Follicular lymphomas can be induced to present alloantigen efficiently: a conceptual model to improve their tumor immunogenicity. Proc. Natl. Acad. Sci. USA 92:8200.[Abstract/Free Full Text]
23. Schultze, J. L., M. J. Seamon, S. Michalak, J. G. Gribben, L. M. Nadler. 1997. Autologous tumor infiltrating T cells cytotoxic for follicular lymphoma cells can be expanded in vitro. Blood 89:3806.[Abstract/Free Full Text]
24. Cardoso, A. A., M. J. Seamon, H. M. Afonso, P. Ghia, V. A. Boussiotis, G. J. Freeman, J. G. Gribben, S. E. Sallan, L. M. Nadler. 1997. Ex vivo generation of human anti-pre-B leukemia-specific autologous cytolytic T cells. Blood 90:549.[Abstract/Free Full Text]
25. Pellat Deceunynck, C., R. Bataille, N. Robillard, J. L. Harousseau, M. J. Rapp, N. Juge Morineau, J. Wijdenes, M. Amiot. 1994. Expression of CD28 and CD40 in human myeloma cells: a comparative study with normal plasma cells. Blood 84:2597.[Abstract/Free Full Text]
26. Zhang, X. G., D. Olive, J. Devos, C. Rebouissou, M. Ghiotto-Ragueneau, M. Ferlin, B. Klein. 1998. Malignant plasma cell lines express a functional CD28 molecule. Leukemia 12:610.[Medline]
27. Robillard, N., G. Jego, C. Pellat-Deceunynck, D. Pineau, D. Puthier, M. P. Mellerin, S. Barille, M. J. Rapp, J. L. Harousseau, M. Amiot, R. Bataille. 1998. CD28, a marker associated with tumoral expansion in multiple myeloma. Clin. Cancer Res. 4:1521.[Abstract]
28. Pellat-Deceunynck, C., M. Amiot, N. Robillard, J. Wijdenes, R. Bataille. 1996. CD11a-CD18 and CD102 interactions mediate human myeloma cell growth arrest induced by CD40 stimulation. Cancer Res. 56:1909.[Abstract/Free Full Text]
29. Zhang, X. G., J. P. Gaillard, N. Robillard, Z. Y. Lu, Z. J. Gu, M. Jourdan, J. M. Boiron, R. Bataille, B. Klein. 1994. Reproducible obtaining of human myeloma cell lines as a model for tumor stem cell study in human multiple myeloma. Blood. 83:3654.[Abstract/Free Full Text]
30. Westendorf, J. J., G. J. Ahmann, R. J. Armitage, M. K. Spriggs, J. A. Lust, P. R. Greipp, J. A. Katzmann, D. F. Jelinek. 1994. CD40 Expression in malignant plasma cells—role in stimulation of autocrine IL-6 secretion by a human myeloma cell line. J. Immunol. 152:117.[Abstract]
31. Yi, Q., S. Dabadghao, A. Osterborg, S. Bergenbrant, G. Holm. 1997. Myeloma bone marrow plasma cells: evidence for their capacity as antigen-presenting cells. Blood 90:1960.[Abstract/Free Full Text]
32. Urashima, M., D. Chauhan, H. Uchiyama, G. J. Freeman, K. C. Anderson. 1995. CD40 ligand triggered interleukin-6 secretion in multiple myeloma. Blood 85:1903.[Abstract/Free Full Text]
33. Bergamo, A., R. Bataille, C. Pellat-Deceunynck. 1997. CD40 and CD95 induce programmed cell death in the human myeloma cell line XG2. Br. J. Haematol. 97:652.[Medline]
34. Pellat-Deceunynk, C., M. Amiot, R. Bataille, I. Van Riet, B. Van Camp, P. Omede, M. Boccadoro. 1995. Human myeloma cell lines as a tool for studying the biology of multiple myeloma: a reappraisal 18 years after. Blood 86:4001.[Free Full Text]
35. Wendtner, C. M., A. Nolte, E. Mangold, R. Buhmann, G. Maass, J. A. Chiorini, E. L. Winnacker, B. Emmerich, R. M. Kotin, M. Hallek. 1997. Gene transfer of the costimulatory molecules B7-1 and B7-2 into human multiple myeloma cells by recombinant adeno-associated virus enhances the cytolytic T cell response. Gene Ther. 4:726.[Medline]
36. Geldhof, A. B., G. Raes, M. Bakkus, S. Devos, K. Thielemans, P. De Baetselier. 1995. Expression of B7-1 by highly metastatic mouse T lymphomas induces optimal natural killer cell-mediated cytotoxicity. Cancer Res. 55:2730.[Abstract/Free Full Text]
37. Yeh, K. Y., B. A. Pulaski, M. L. Woods, A. J. McAdam, A. A. Gaspari, J. G. Frelinger, E. M. Lord. 1995. B7-1 enhances natural killer cell-mediated cytotoxicity and inhibits tumor growth of a poorly immunogenic murine carcinoma. Cell Immunol. 165:217.[Medline]
38. Jourdan, M., M. Ferlin, E. Legouffe, M. Horvathova, J. Liautard, J. F. Rossi, J. Wijdenes, J. Brochier, B. Klein. 1998. The myeloma cell antigen syndecan-1 is lost by apoptotic myeloma cells. Br. J. Haematol. 100:637.[Medline]
39. Wijdenes, J., W. C. Vooijs, C. Clement, J. Post, F. Morard, N. Vita, P. Laurent, R. X. Sun, B. Klein, J. M. Dore. 1996. A plasmocyte selective monoclonal Ab (B-B4) recognizes syndecan-1. Br. J. Haematol. 94:318.[Medline]
40. Horvathova, M., J. P. Gaillard, J. Liautard, C. Duperray, T. Lavabre-Bertrand, P. Bourquard, J. F. Rossi, B. Klein, J. Brochier. 1995. Identification of novel and specific antigens of human plasma cells by mAb. S. F. Schlossman, and L. Boumsell, and W. Gilks, and J. M. Harlan, and T. Kishimoto, and C. Morimoto, and J. Ritz, and S. Shaw, and R. Silverstein, and T. Springer, and T. F. Tedder, and R. F. Todd, eds. Leucocyte Typing V 713. Oxford University Press, London.
41. San Miguel, J. F., R. Garcia-Sanz, M. Gonzalez, M. J. Moro, J. M. Hernandez, F. Ortega, D. Borrego, M. Carnero, F. Casanova, R. Jimenez. 1995. A new staging system for multiple myeloma based on the number of S-phase plasma cells. Blood 85:448.[Abstract/Free Full Text]
42. Sun, R. X., Z. Y. Lu, J. Wijdenes, J. Brochier, C. Hertog, J. F. Rossi, B. Klein. 1997. Large scale and clinical grade purification of syndecan-1⁺ malignant plasma cells. J. Immunol. Methods 205:73.[Medline]
43. Rebouissou, C., J. Wijdenes, P. Autissier, K. Tarte, V. Costes, J. Liautard, J. F. Rossi, J. Brochier, B. Klein. 1998. A gp130 interleukin-6 transducer-dependent SCID model of human multiple myeloma. Blood 91:4727.[Abstract/Free Full Text]
44. Tarte, K., Z. Y. Lu, G. Fiol, E. Legouffe, J. F. Rossi, B. Klein. 1997. Generation of virtually pure and potentially proliferating dendritic cells from non-CD34 apheresis cells from patients with multiple myeloma. Blood 90:3482.[Abstract/Free Full Text]
45. Portier, M., X. G. Zhang, E. Caron, Z. Y. Lu, R. Bataille, B. Klein. 1993. -Interferon in multiple myeloma: inhibition of interleukin-6 (IL-6) dependent myeloma cell growth and downregulation of IL-6-receptor expression in vivo. Blood 81:3076.[Abstract/Free Full Text]
46. Urashima, M., G. Teoh, D. Chauhan, Y. Hoshi, A. Ogata, S. P. Treon, R. L. Schlossman, K. C. Anderson. 1997. Interleukin-6 overcomes p21WAF1 upregulation and G1 growth arrest induced by dexamethasone and interferon- in multiple myeloma cells. Blood 90:279.[Abstract/Free Full Text]
47. Ruiz-Arguelles, G. J., J. F. San Miguel. 1994. Cell surface markers in multiple myeloma. Mayo Clin. Proc. 69:684.[Medline]
48. Leo, R., M. Boeker, D. Peest, R. Hein, R. Bartl, J. E. Gessner, J. Selbach, G. Wacker, H. Deicher. 1992. Multiparameter analyses of normal and malignant human plasma cells: CD38⁺⁺, CD56⁺, CD54⁺, cIg⁺ is the common phenotype of myeloma cells. Ann. Hematol. 64:132.[Medline]
49. Anderson, K. C., M. P. Bates, B. Slaughenhoupt, S. F. Schlossman, L. M. Nadler. 1984. A monoclonal Ab with reactivity restricted to normal and neoplastic plasma cells. J. Immunol. 132:3172.[Abstract]
50. Funaro, A., G. C. Spagnoli, C. M. Ausiello, M. Alessio, S. Roggero, D. Delia, M. Zaccolo, F. Malavasi. 1990. Involvement of the multilineage CD38 molecule in a unique pathway of cell activation and proliferation. J. Immunol. 145:2390.[Abstract]
51. Costes, V., V. Magen, E. Legouffe, L. Durand, P. Baldet, J. F. Rossi, B. Klein, and J. Brochier. 1999. The MI15 monoclonal Ab (anti-syndecan-1) is a reliable marker for quantifying plasma cells in paraffin-embedded bone marrow biopsies. Hum. Pathol. In press.
52. Jourdan, M., K. Tarte, E. Legouffe, J. Brochier, J. F. Rossi, B. Klein. 1999. Tumor necrosis factor is a survival and proliferation factor for human myeloma cells. Eur. Cytokine. Netw. 10:65.[Medline]
53. Spets, H., P. Georgii-Hemming, J. Siljason, K. Nilsson, H. Jernberg-Wiklund. 1998. Fas/APO-1 (CD95)-mediated apoptosis is activated by interferon- and interleukin-6 (IL-6)-dependent and IL-6-independent multiple myeloma cell lines. Blood 92:2914.[Abstract/Free Full Text]
54. Ranheim, E. A., T. J. Kipps. 1993. Activated T cells induce expression of B7/BB1 on normal or leukemic B cells through a CD40-dependent signal. J. Exp. Med. 177:925.[Abstract/Free Full Text]
55. Cella, M., D. Scheidegger, K. Palmer Lehmann, P. Lane, A. Lanzavecchia, G. Alber. 1996. Ligation of CD40 on dendritic cells triggers production of high levels of interleukin-12 and enhances T cell stimulatory capacity: T-T help via APC activation. J. Exp. Med. 184:747.[Abstract/Free Full Text]
56. Kiener, P. A., P. Moran-Davis, B. M. Rankin, A. F. Wahl, A. Aruffo, D. Hollenbaugh. 1995. Stimulation of CD40 with purified soluble gp39 induces proinflammatory responses in human monocytes. J. Immunol. 155:4917.[Abstract]
57. Alderson, M. R., R. J. Armitage, T. W. Tough, L. Strockbine, W. C. Fanslow, M. K. Spriggs. 1993. CD40 expression by human monocytes: regulation by cytokines and activation of monocytes by the ligand for CD40. J. Exp. Med. 178:669.[Abstract/Free Full Text]
58. Prince, H. M., S. Dessureault, S. Gallinger, M. Krajden, D. R. Sutherland, C. Addison, Y. Zhang, F. L. Graham, A. K. Stewart. 1998. Efficient adenovirus-mediated gene expression in malignant human plasma cells: relative lymphoid cell resistance. Exp. Hematol. 26:27.[Medline]
59. Stewart, A. K., A. D. Schimmer, D. J. Bailey, I. D. Dube, D. Cappe, R. C. Moen, J. Gauldie, F. L. Graham. 1998. In vivo adenoviral-mediated gene transfer of interleukin-2 in cutaneous plasmacytoma. Blood 91:1095.[Free Full Text]
60. Pfeiffer, S., R. P. Gooding, J. F. Apperley, H. Goldschmidt, D. Samson. 1997. Dendritic cells generated from the blood of patients with multiple myeloma are phenotypically and functionally identical to those similarly produced from healthy donors. Br. J. Haematol. 98:973.[Medline]
61. Dabadghao, S., S. Bergenbrant, D. Anton, W. He, G. Holm, Q. Yi. 1998. Anti-idiotypic T-cell activation in multiple myeloma induced by M-component fragments presented by dendritic cells. Br. J. Haematol. 100:647.[Medline]

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