Generation of potent Th1 responses from patients with lymphoid malignancies after differentiation of B lymphocytes into dendritic-like cells

Mohamad Mohty1, Daniel Isnardon1, Aude Charbonnier1, Marina Lafage-Pochitaloff2, Michele Merlin3, Danielle Sainty4, Daniel Olive5 and Béatrice Gaugler5

1 Laboratoire d’Immunologie des Tumeurs, 2 Laboratoire de Cytogénétique, 3 Laboratoire de Biochimie, 4 Laboratoire d’Hématologie, Institut Paoli-Calmettes, Université de la Méditerranée, 232 Boulevard Ste Marguerite, 13273 Marseille Cedex, France 5 INSERM U119, 13009 Marseille, France

Correspondence to: B. Gaugler; E-mail: gauglerb{at}marseille.fnclcc.fr
Transmitting editor: L. Moretta


    Abstract
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 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
Dendritic cells (DC) are a group of potent antigen-presenting cells (APC) specialized for initiating T cell immune responses. They originate from the bone marrow and upon stimulation with bacterial products, cytokines or CD40 ligation they acquire the ability to migrate to the secondary lymphoid organs. In vitro DC can be generated from human CD34+ bone marrow cells and CD14+ peripheral blood monocytes after culture with different cytokine combinations. Since most leukemic cells and tumors in general are devoid of APC capacities, various strategies have been used to increase their recognition and confer the capacity of antigen presentation on them. Because of our interest in the design of vaccine immunotherapy protocols for the adjuvant treatment of patients with lymphoid malignancies (LM), we chose to explore the capacity of human acute lymphoblastic leukemia, chronic lymphocytic leukemia and plasma cell leukemia to differentiate into cells with APC and DC features. Our results among a sample of 10 patients demonstrate that such approach is feasible. Leukemic cells could be induced in the presence of IL-4 and CD40L to exhibit a DC morphology with a phenotype of mature DC-like cells. They could also induce a potent proliferative response in naive CD4+ T cells. In addition, they expressed chemokine receptor CCR7 and CD62L, and could drive T cells towards a Th1 response with secretion of IFN-{gamma}. Our strategy leading to increased LM cell immunogenicity may have potential clinical applications and LM appear to be attracting candidates for adjuvant vaccination and adoptive immunotherapy.

Keywords: antigen-presenting cell, dendritic cell, immunogenicity, lymphoid malignancy, Th1 response


    Introduction
 Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
Dendritic cells (DC) are a system of potent antigen-presenting cells (APC) specialized for initiating primary T cell immune responses (1). DC are considered as important elements in the induction of specific antitumor immune responses. DC ultimately derive from hematopoietic precursors, although little is known about their lineage of origin. They can be generated in vitro from CD34 cord blood or bone marrow progenitors in the presence of granulocyte macrophage colony stimulating factor (GM-CSF) and tumor necrosis factor (TNF)-{alpha}, as well as from peripheral blood monocytes in the presence of GM-CSF and IL-4 (2,3).

Since most leukemic cells and tumors in general are devoid of APC capacities, various strategies have been used to increase their recognition. Transfection of co-stimulatory molecules can render non-immunogenic malignant cells immunogenic (4). An alternative approach was the fusion of malignant cells with B cells or DC (57). All these approaches resulted in induction of cytotoxic T lymphocyte (CTL)-mediated protective antitumor immunity. It has been previously shown that activation of follicular lymphoma cells or pre-B cell leukemias via CD40 could induce or up-regulate both adhesion and B7 co-stimulatory molecules (8,9). Moreover, using the CD40-stimulation strategy, autologous antileukemia-specific CTL could be generated in some patients with pre-B cell leukemias (10). Another promising approach would be the differentiation of tumoral cells themselves into DC or at least into cells with APC functions. Such cells are expected to combine both APC function and expression of tumor antigens. Recently, hematopoietic tumors appeared to be candidates for DC differentiation. Several studies, including one from our group, succeeded in in vitro DC differentiation of leukemic blasts, derived from chronic myeloid leukemia or acute myeloid leukemia (1118). Except for the study of Cignetti et al. (16) who described the generation of DC like cells from two patients with CD34+ acute lymphoblastic leukemia (ALL), without detailed functional characterization, all previous reports concerned leukemic cells belonging to the myeloid lineage.

In the present report, we investigated whether CD19+ B lymphocytes could be induced to differentiate into dendritic-like cells (B-DC). We show that in the presence of IL-4 and CD40L, human CD19+ B lymphocytes can convert after culture in vitro into cells with mature DC features demonstrated by their typical morphology, their phenotype and their powerful capacity to induce naive CD4 allogeneic T cell proliferation towards a Th1 response profile. Because of our interest in the design of vaccine immunotherapy protocols for the adjuvant treatment of patients with lymphoid malignancies (LM), we then chose to explore the capacity of human ALL, chronic lymphocytic leukemia (CLL) and plasma cell leukemia (PCL) to differentiate into B-DC. Our results among a sample of 10 LM demonstrate that such approach is feasible and make them potential candidates for adjuvant vaccination therapies.


    Methods
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 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
Blood samples
Peripheral blood mononuclear cells (PBMC) from healthy donors (Regional transfusion center, Marseille, France) and from patients were isolated on Ficoll-Hypaque (Pharmacia, Uppsala, Sweden) gradients prior to cryo-preservation. Blood samples from patients with LM were obtained after informed consent at diagnosis and before any chemotherapy according to institutional guidelines. Relevant clinical and diagnostic laboratory data for all cases are shown in Table 1. All patients were diagnosed and/or treated at the Institut Paoli-Calmettes (Marseille, France) between 1993 and 2000.


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Table 1. Patient characteristics
 
Cell lines
Murine L cells transfected with human CD40L were kindly provided by Schering-Plough (Laboratory for Immunological Research, Dardilly, France) (19) and used after a 75 Gy irradiation.

Cell separation and DC generation
CD14+ monocytes were immunomagnetically purified with CD14 mAb-conjugated microbeads. (Miltenyi Biotec, Bergisch Gladbach, Germany). Purification of CD19+ B lymphocytes was performed by incubation of PBMC with anti-CD19 (Diaclone, Besançon, France) followed by positive selection using goat anti-mouse conjugated microbeads (Miltenyi Biotec). Purity of the CD14+ and CD19+ cells by flow cytometry analysis was always >98%. Culture experiments were performed in RPMI 1640 medium containing 10% FCS (Biowhittaker, Verviers, Belgium) in the presence of 100 ng/ml GM-CSF (kind gift of Novartis, Berne, Switzerland) and 20 ng/ml IL-4 (kind gift of Schering-Plough Research Institute, Kenilworth, NJ) for Mo-DC generation. On day 6, final maturation of Mo-DC was induced by adding 75-Gy-irradiated CD40L-transfected L cells (2 x 105/well). For B-DC generation, CD19+ cells or leukemic cells which had no proliferative capacity were cultured in the presence of 20 ng/ml IL-4 and 75-Gy-irradiated CD40L-transfected cells (2 x 105/well). The medium was replenished with cytokines every 3 days.

T cell separation
CD4+/CD45RA+ naive T cells were purified by negative selection of adult blood PBMC using goat anti-mouse Ig-coated magnetic beads (Beckman-Coulter, Marseille, France) incubated with mAb against CD8, CD14, CD56 (D. Olive, INSERM U119), CD19 (Diaclone) and CD45RO (Beckman-Coulter). Purity was >99% as controlled by FACS analysis.

Flow cytometry analysis
The following mAb (clone names) were used in this study for flow cytometry: CD1a (BL6), CD4 (13B8.2), CD5 (BL1a), CD13 (Immu103.44), CD14 (RMO52), CD19 (J4.119), CD33 (D3HL60.251), CD40 (MAB89), CD54 (84H10), CD58 (AICD58), CD62L (DREG56), CD80 (MAB104), CD83 (HB15a), CD116 (SC06), anti-HLA-DR (Immu-357), anti-mouse IgG1(679.1Mc7), anti-mouse IgG2a (U7.27), anti-mouse IgG2b (MOPC-195) and anti-mouse IgM (GC323) from Coulter-Immunotech (Marseille, France). CD86 (IT2.2), CD123 (9F5), CCR5 (2D7) and CCR7 (2H4) were purchased from PharMingen (San Diego, CA). All mAb were used as FITC-, phycoerythrin-, Cy5- or allophycocyanin-conjugated mAb, or with phycoerythrin-conjugated F(ab')2-fragments of goat anti-mouse IgM antibody when using CCR7 mAb. Samples were analyzed using a FACSCalibur (BD Biosciences, Le Pont de Claix, France). Data for at least 10 x 103 cells/sample were acquired and analyzed using CellQuest software (BD Biosciences).

FITC–dextran capture analysis
To assess endocytosis of Mo-DC and B-DC, FITC–dextran (Sigma, St Quentin Fallavier, France) was used according to the method described previously (2). Briefly, the cells were incubated with 0.1 mg/ml FITC–dextran at 37°C for 1 h. The results were analyzed as mean fluorescence intensity after subtracting the background in which cells were incubated with FITC–dextran at 4°C.

Confocal microscopy
Cells were adhered to polylysine-coated glass slides for 30 min at room temperature, fixed in 4% paraformaldehyde and permeabilized in 0.1% Triton in PBS. Cells were then labeled with different primary mAb and revealed by species-specific Alexa 488-, TRITC- (Molecular Probes, Eugene, OR) or Cy5-labeled secondary antibodies (Jackson ImmunoResearch, West Baltimore Pike, PA). Slides were then mounted using fluorescent mounting medium (Dako, Trappes, France). Confocal analysis was performed with a TCS NT microscope equipped with argon and krypton ion lasers and a x100 1.3NA PL fluotar objective (Leica Microsystem, Heidelberg, Germany).

Fluorescence in situ hybridization analysis (FISH)
Interphase FISH was performed as previously described on cytospin preparations with sorted CD19+/CD83+ cells from patients selected for detection of their cytogenetic abnormality (20). For detection of t(9;22), the LSI bcrabl ES probe set (Vysis, Downers Grove, IL) was used to examine interphase nuclei. Normal cells were expected to display randomly distributed, two red (bcr gene) and two green (abl gene) hybridization signals. As the bcr and abl probes encompass the mbcr and abl breakpoints respectively, leukemic mbcr+ cells were expected to display a red, a green and two fusion signals. At least 200 nuclei were examined in each sample under fluorescence microscopy by two independent observers to quantify the percentage of cells bearing the bcrabl translocation.

Primary mixed lymphocyte reaction (MLR)
Serial dilutions (30 x 103–102 cells/well) of 25-Gy-irradiated stimulating cells were cultured in triplicate with 105 allogeneic naive T cells in 96-well flat bottom plates (Costar, Corning NY). Proliferation of T cells was monitored by measuring [methyl-3H]thymidine (1 µCi/well; Amersham, Little Chalfont, UK) incorporation during the last 16 h of a 6-day culture. Thymidine uptake was counted on a gas-phase ß-counter (Matrix 9600; Packard, Downers Grove, IL). Cultures were maintained in a humidified atmosphere at 37°C and 5% CO2. As stimulating cells, freshly isolated monocytes, B lymphocytes, leukemic blasts, immature Mo-DC, matured Mo-DC and B-DC were used.

Cytokine production assay
Allogeneic naive CD4+/CD45RA+ T cells were co-cultured with freshly isolated monocytes, B lymphocytes, leukemic blasts, immature Mo-DC, matured Mo-DC and B-DC. Cells were harvested after 6 days, and replated in 48-well culture plates at 5 x 105 cells/well in the presence of phorbol myristate acetate (Sigma; 25 ng/ml) and ionomycin (Sigma; 1 µg/ml). After 48 h supernatants were harvested and frozen until analysis. Cytokines were analyzed by ELISA using the OptEIA set for IFN-{gamma} and IL-10 (BD Biosciences).

Intracellular analysis of cytokine production
Anti-IL-4–FITC, anti-IL-10–phycoerythrin, anti-IFN-{gamma}–allophycocyanin and FITC/phycoerythrin/allophycocyanin-conjugated isotypic mAb (PharMingen) were used according to the manufacturer’s instruction. In brief, 6 days after stimulation, T cells were activated with phorbol myristate acetate and ionomycin for 6 h in the presence of 10 µg/ml of Brefeldin A (Sigma). Cells were collected, washed, fixed and permeabilized using the CytoStain Kit (PharMingen), and stained with 0.5 µg/test of cytokine-specific mAb.


    Results
 Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
In vitro generation of B-DC from B lymphocytes
Normal CD14+ peripheral blood monocytes can be induced to differentiate into immature Mo-DC by GM-CSF and IL-4. Immature Mo-DC can acquire a mature phenotype following exposure to TNF-{alpha}, CD40L or other maturation agents like lipopolysaccharides. We assessed whether CD19+ B lymphocytes can be similarly induced to differentiate into cells with DC features. CD19+ B cells were sorted by immunomagnetic selection from PBMC from different healthy donors and cultured in the presence of various concentrations of GM-CSF, IL-3, IL-4 or in combination with CD40L. A combination of IL-4 and CD40L was found to be optimal for generation of B cells with DC morphology in terms of viability, yield and phenotype. Moreover, the use of CD40L was mandatory since IL-4 alone did not induce any DC features on B cells (data not shown). Therefore, this combination was subsequently used for phenotypic and functional characterization. Before culture, freshly isolated CD19+ B cells appeared as dispersed, small spherical cells with a smooth surface morphology (data not shown). After 3–4 days of culture, B cells appeared larger and were associated in non-adherent grape-like clusters with short projections emerging from the surface. Between days 5 and 8, like maturing Mo-DC, CD19+ cells displayed dramatic morphological changes with size increase. In clusters or dispersed, non-adherent cells with large cell bodies and long dendritic projections were the predominant population (Fig. 1A and D).



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Fig. 1. Morphology and phenotype of B-DC and Mo-DC. Two-color immunofluorescence confocal microscopy of HLA-DR and CD83 was performed on Mo-DC (A, B and C) and on B-DC cultured for 8 days with GM-CSF, IL-4 plus CD40L for the last 2 days or IL-4 and CD40L respectively (D, E and F). Interferential contrast transmission (x100) view is shown in (A) and (D), CD83 in (B) and (E), and HLA-DR in (C) and (F). Results are representative of four independent experiments.

 
Next, we examined the capacity of B cells differentiated in the presence of IL-4 and CD40L for endocytosis by uptake of FITC–dextran. Like mature Mo-DC, B cells with DC morphology showed very little, if any, FITC–dextran uptake (data not shown). Hence, CD19+ B cells could be induced to display in vitro morphological and endocytic characteristics similar to mature Mo-DC. Such cultured B cells with DC-like characteristics will be referred to as B-DC hereinafter.

On the phenotypic level, before culture, CD19+ cells were negative for CD1a, CD80, CD83 and CD86, negative or dimly positive for CD54 and CD58, but positive for CD40, HLA-DR and HLA class I (data not shown). After culture, we investigated the expression of CD83 which is classically found on peripheral mature DC (21). For all normal donors, a CD83 expression could be obtained in mature Mo-DC after 2 days exposure to CD40L, as well as on all CD19+ cells after 5–6 days of culture in the presence of IL-4 and CD40L (Fig. 2A and B). Acquisition of CD83 on B-DC and mature Mo-DC was further confirmed by confocal microscopy staining (Fig. 1B and E). As expected, the monocytic marker CD14 was down-regulated on all of immature and mature Mo-DC, but B cells never expressed CD14 (Fig. 2A and B). CD1a, another lineage marker of DC, was expressed on Mo-DC, but never found on B-DC. We next analyzed on the CD19+/CD83+ B-DC fraction the expression of the adhesion molecules (CD54 and CD58), MHC molecules (MHC class I and HLA-DR) and co-stimulatory molecules (CD40, CD80 and CD86). All these molecules were up-regulated (CD40, CD54, CD58, MHC class I and HLA-DR) or induced (CD80 and CD86) (Fig. 2A and B). Confocal staining of B-DC indicated as for mature Mo-DC, a large expression and peripheral distribution of HLA-DR molecules comparable to that obtained in mature Mo-DC (Fig. 1C and F). B-DC did not express the myeloid markers CD13 and CD33. Mo-DC expressed GM-CSF receptor {alpha} (CD116), which was not or dimly expressed on B-DC. B-DC did not express IL-3 receptor {alpha} (CD123), which is a classical marker of the so-called plasmacytoid DC (19) (Fig. 2A and B).



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Fig. 2. Phenotype of Mo-DC and B-DC. Mo-DC were obtained by culturing CD14+ monocytes for 6 days in the presence of GM-CSF and IL-4 followed by 48 h in the presence of CD40L (A). B lymphocytes were cultured for 8 days with IL-4 and CD40L (B). Cells were analyzed by flow cytometry for the expression of the indicated mAb (black histograms). Staining with isotypic control is shown (white histograms). Results are representative of at least 10 experiments with different donors.

 
To investigate their allo-stimulatory function, B-DC generated after 6–8 days of culture were used to stimulate naive CD4+/CD45RA+ T cells from an unrelated normal donor at different stimulator:responder ratios. B-DC were found to be potent allogeneic MLR stimulators. At all stimulator:responder ratios, the stimulating activity of B-DC was as powerful as that of mature Mo-DC. Furthermore, in comparison with immature Mo-DC, the stimulating activity of B-DC was 2-fold higher. In the same experiments, freshly isolated peripheral blood CD19+ B cells and resting monocytes did not induce any proliferation of naive allogeneic CD4+ T cells (Fig. 3). Thus, potent and sustained proliferation of naive CD4+ T cells is another prominent feature of B-DC.



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Fig. 3. Allo-stimulatory capacity of B-DC. Proliferation of allogeneic CD4+ naive T lymphocytes was measured in response to various numbers of irradiated stimulating cells. The mean results obtained from three different donors are indicated. Freshly isolated monocytes and B lymphocytes were used as controls.

 
In vitro generation of leukemic B-DC from patients with LM
Due to the lack of ability of tumors to behave as APC, presentation of tumor antigens in vivo would proceed mainly by cross-priming with capture and processing of tumor antigens by DC (22). Thus, we investigated whether malignant B cells such as in ALL, and other LM such as CLL and PCL could also be inducible into B-DC. Leukemic cells positive for the B cell marker CD19 and negative for the monocytic marker CD14 (except for patient LM177 with a 14% population of CD14+ cells), from 10 patients with various LM (Table 1) were cultured in the presence of IL-4 and CD40L. As soon as 4–5 days of culture, cells consistently displayed an increase in cell size and showed cell clusters with dendritic morphology (Fig. 4A). Before culture, leukemic cells were CD1a, CD80, CD83 and CD86 negative, but were CD40, HLA class I and HLA-DR positive (data not shown). After culture, among the 10 cases, CD83+ cells reached a mean of 40% (SD 22%; range 5–76% ). Interestingly, although malignant B cells were heterogeneous, ranging from B cell progenitors/precursors to plasma cells, a significant percentage of malignant B-DC could always be obtained. These CD83+ cells remained CD19+. CD1a expression remained negative, but we observed an important up-regulation of CD40, CD54, CD58, HLA class I and HLA-DR molecules. Also, leukemic cells acquired co-stimulatory molecules CD80 and CD86 (Fig. 5). Acquisition and distribution of CD83 and HLA-DR molecules were confirmed by confocal microscopy staining (Fig. 4B and C).



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Fig. 4. Morphology and cytogenetic analysis of in vitro differentiated leukemic blasts. Leukemic cells from patient LM8 were cultured for 6 days with IL-4 and CD40L, and analyzed by confocal microscopy. Interferential contrast transmission (x100) view (A). CD83 mAb staining (B). HLA-DR mAb staining (C). Results are representative of four experiments with different patients. The leukemic origin was determined by FISH experiments with a specific probe for bcrabl (patient LM274) (D). FISH analysis was performed on purified CD19+/CD83+ cells from patients LM4 and LM274.

 


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Fig. 5. Phenotype of leukemic cells from leukemic patients after in vitro culture. Leukemic cells from patient LM8 were stained after 8 days of culture with the indicated mAb and analyzed by flow cytometry. Phenotypic analysis was performed on the 10 patients included in this study.

 
Leukemic origin of leukemia-derived B-DC
The leukemic origin of CD19+/CD83+ cells arising after culture was investigated by FISH experiments. Purified CD19+/CD83+ cells from patients LM4 and LM274 were analyzed for the persistence of the initial bcrabl fusion gene. After culture, the percent of CD83+ cells that were positive for bcrabl was comparable to the percent of unfractionated blasts that were positive for bcrabl. An example from LM4 bearing the bcrabl fusion gene is depicted in Fig. 4(D). In addition, a unique property of B lymphocytes in CLL is the presence of the CD5 molecule (23). The expression of CD5 on CLL B cells is so unique that many consider its absence to argue against a diagnosis of B cell CLL (24). Thus, the expression of CD5 (Fig. 5) on the whole culture-differentiated leukemic cells in both patients with CLL in our series (LM8 and LM283) further confirmed the leukemic origin of DC-like cells obtained by differentiation of LM cells in vitro.

Profile of chemokine receptor expression on leukemic B-DC
As a way to understand the regulation of leukemic B-DC traffic, we examined the chemokine receptor expression on their surface in comparison with immature and mature Mo-DC. Flow cytometric analysis revealed, as previously described (25), that immature Mo-DC expressed CCR5, which was down-regulated upon maturation. In contrast, mature Mo-DC were found to express CCR7, which is usually induced on mature Mo-DC (25). Normal and leukemic B-DC did not express CCR5 at any stage of their differentiation process, but could acquire the expression of CCR7. In addition, B-DC, but not Mo-DC, expressed CD62L (L-selectin), a molecule which is known to mediate adhesion and ‘rolling’ on high endothelial venules (HEV) (26) (Table 2).


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Table 2. Chemokine receptor and CD62L expression on Mo-DC and B-DC
 
CD4+ naive T cells proliferative response in the presence of leukemic B-DC
The stimulatory activity for allogeneic naive CD4+/CD45RA+ T cells in MLR assay was compared among the different normal and leukemic DC fractions as shown in Fig. 6(A). Leukemic B-DC showed a potent allo-stimulatory potential as compared to B-DC generated from healthy volunteers. The leukemic B-DC elicited higher proliferation than did the fresh/uncultured leukemic population. Among the six cases tested in allogeneic MLR, the proliferation induced was at least 3-fold higher than the magnitude of that induced by freshly isolated leukemic cells (Fig. 6B) irrespective of the stimulator:responder ratio in culture (data not shown). Interestingly, the least potent allo-stimulatory effect (which was still greater than that of fresh leukemic cells) was obtained with blasts from patient LM240, who had the lowest percentage of B-DC after culture (Fig. 6B). Thus, the superior stimulatory activity of cultured leukemic B cells compared with the freshly isolated blasts was established.



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Fig. 6. Proliferative response of allogeneic CD4+ naive T lymphocytes stimulated by leukemic cells. Graded numbers of mature Mo-DC (filled circles), in vitro-differentiated B-DC from healthy donors (filled triangles) or leukemic blasts from patients (filled squares) were co-cultured with 105 naive CD4+ T cells for 6 days. Freshly isolated monocytes (open circles), B lymphocytes from a healthy donor (open triangles) or fresh leukemic blasts (open squares) were used as controls (A). Allo-stimulatory capacity of tumor cells from LM patients. In vitro differentiated (black histograms) or freshly isolated (open histograms) leukemic cells were cultured with 105 allogeneic naive CD4+ T cells for 6 days (B). The ratio between the proliferative response measured by [3H]thymidine incorporation of 105 T cells induced by 12 x 103 leukemic or normal B lymphocytes and mature Mo-DC is represented. Results are represented as the mean of the ratio obtained from three experiments performed with T cells isolated from three different healthy donors.

 
Th1 polarization capacity of B-DC
We next examined the nature of primary allogeneic T cell responses induced by normal and leukemic B-DC. Naive CD4+ CD45RA+ T cells isolated from human peripheral blood were co-cultured for 7 days with immature Mo-DC, mature Mo-DC, normal B-DC and leukemic B-DC generated from six patients in our series. The cultured cells were counted and re-stimulated with phorbol myristate acetate and ionomycin for either 5 h for single-cell cytokine analyses by flow cytometry (Fig. 7A) or 48 h for cytokine secretion analyses (Fig. 7B). T cells originally cultured with normal and leukemic B-DC secreted IFN-{gamma} (Fig. 7B), but little or undetectable IL-4 and IL-10. T cells cultured with mature Mo-DC secreted the highest amounts of IFN-{gamma} (1-fold higher) (Fig. 7B). These polarized cytokine production profiles were confirmed by single-cell cytokine analysis using intracellular immunofluorescence flow cytometry (Fig. 7A). Thus, leukemic B-DC can drive naive T cells towards a Th1 response profile.



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Fig. 7. Th1-polarizing capacity of in vitro differentiated leukemic cells. Production of IFN-{gamma}, IL-4 and IL-10 was measured by intracellular staining of CD4+ T cells stimulated with the in vitro differentiated leukemic cells (patient LM90) and B lymphocytes from a healthy donor (A). Results are representative of six experiments with different patients and healthy donors. IFN-{gamma} content in the supernatant of co-culture of T cells with the indicated stimulating cells was measured by ELISA (B). Results are represented as the mean + SD obtained from six patients and three different healthy donors.

 

    Discussion
 Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
In the present report, we have demonstrated that peripheral blood CD19+ B lymphocytes and leukemic B cells could be induced in the presence of IL-4 and CD40L to differentiate into cells with DC features. B-DC exhibited DC morphology, had a phenotype of mature APC and could induce a potent proliferative response in naive CD4+ T cells. All these features are in accordance with DC properties currently accepted for the characterization of DC subsets (2729). In addition, B-DC expressed chemokine receptor CCR7 and CD62L, and could drive T cells towards a Th1 response with secretion of IFN-{gamma}. These findings raise the question whether CD19+ B cells could have a B/DC bipotential lineage capacity. DC were originally thought to be only of myeloid origin. However, different observations indicated that DC might be of diverse origins (30). Recent investigations showed that early T lineage cells can differentiate into B cells, NK cells and DC (31). In this respect, Galy et al. showed that a multipotential hematopoietic stem cell can give rise to lymphocytes, NK cells and DC (32). In addition, B lymphocyte lineage cell lines can convert to macrophage-like cells spontaneously or through experimental manipulation (33). Using a murine model, CD19+ pro-B cells were shown to be able to give rise to DC in vitro (34). In a large study by Shultze et al., CD40-activated B cells were shown to be efficient APC to generate autologous antigen-specific T cells (35). Moreover, in an attempt to generate a novel anti-B cell mAb, Zhong et al. isolated a B lymphocyte subset with DC morphology and efficient APC function in the first 24 h after isolation (36). On the pathological level, although malignancies involving DC are rare, some Hodgkin’s lymphomas can have DC features and phenotypic markers (37). Furthermore, some malignant B cells from non-Hodgkin lymphomas were found to be able to express functional co-stimulatory molecules (CD80 and CD86) and to be fully competent APC (38). Taken together, all these findings suggest a close relationship between DC and B lymphocytes.

Our results show that the simultaneous association of IL-4 and CD40L can induce normal and leukemic B cells towards cells with striking DC features. Although CD40L was already proved to induce proliferation and up-regulation of co-stimulatory molecules on normal and malignant B cells (20,39,40), the addition of IL-4 was mandatory to obtain the highest yields of fully functional B-DC. Furthermore, the role of IL-4 is essential since B lymphocytes triggered through their CD40 differentiate into plasma cells in response to IL-10, but not IL-4 (41). Moreover, signaling through CD40 can abrogate the tolerogenic capacity which has been sometimes associated with resting B lymphocytes (42) and make them therefore potential adjuvants for immunotherapy. In our experiments, although we did not distinguish between peripheral blood naive and memory B cells for B-DC generation, our culture system did not direct differentiation either towards plasma cells, or secretion of Ig (data not shown), but induced morphologic, phenotypic and functional changes. These properties, including the expression of the CD83 DC-associated marker, might help to define the maturation pattern of these cells towards the DC lineage. The crucial role of co-stimulatory molecules in the generation of an antileukemic response has been shown in murine leukemia (44,45). Along with the acquisition of adhesion and co-stimulatory molecules, the immune response requires a timely interaction among different cell types within distinct microenvironments. The migration of DC from the tumor site to the secondary lymphoid organs is believed to be one of the critical events (46). CCR7 is an important player in the mechanism by which T lymphocytes and DC enter secondary lymphoid organs through HEV. In the same manner, CD62L is known to mediate adhesion and ‘rolling’ on HEV (26). Indeed CD62L is expressed at high levels by naive T cells that reach the lymph node through HEV. In this study, leukemic B-DC expressed CCR7 and CD62L. After injection in vivo, it could therefore be hypothesized that a high proportion of these cells would be trapped in T cell areas of lymph nodes. B-DC never expressed CCR5, a chemokine receptor which favors the redirection of DC into inflammatory and tumor sites (47). This finding is in accordance with the absence of detection of an immature state in B-DC which were also devoid of dextran endocytic activity. Therefore, for optimal efficiency, one could assume that B-DC must already retain at least some leukemia-related proteins, avoiding the antigen capture step and homing directly into lymph nodes where they would be able to initiate an efficient antitumor immune response. In this respect, a critical parameter is to retain part or all of the tumoral antigens during in vitro differentiation. FISH analysis and phenotypic markers confirmed that malignant B-DC in our study still retain the same cytogenetic abnormalities and phenotypic markers expressed by freshly isolated leukemic cells. Therefore, B-DC in leukemic patients may retain at least some features of the malignant clone, like some leukemia-related proteins associated with the cytogenetic abnormality. Thus, we can assume that leukemic B-DC, while directing a Th1 response profile, will help in generating antileukemic cytotoxic responses better than fresh tumoral cells. It has been already demonstrated that the immune balance (Th1/Th2 balance) controlled by cytokines produced by Th1 and Th2 cells plays an important role in immunoregulation, including antitumor immunity (48). Th1 and Th2 cells are cross-regulatory in vitro, and the balance of these cells in vivo determines the character of cell-mediated immune and inflammatory responses (49). The Th1 cells that produce IFN-{gamma} have been shown to exert a powerful antitumor effect, whereas a Th2 profile may have an opposite effect, i.e. down-regulation of innate and acquired antitumor immunity (50). Our strategy leading to induced or increased LM cell immunogenicity while acquiring essential chemokine receptors for trafficking into secondary lymphoid organs may have potential clinical applications like vaccination in vivo to generate antileukemia cytotoxic T effectors or identification of LM tumor antigens. Moreover, leukemic B-DC might be used in vitro for activating antileukemic T cells for use in an allogeneic or autologous setting. Therefore DC-like cells generated from patients with LM, while driving a potent Th1 profile, appear to be attractive candidates for adjuvant vaccination and adoptive immunotherapy.


    Acknowledgements
 
This study was supported by a grant from the ‘Fondation de France’ (to M. M.) and from the ‘Société Française de Greffe de Moelle et de Thérapie Cellulaire’ (to M. M.). We thank R. Galindeau for assistance in cell sorting; S. Just-Landi and N. Baratier for excellent technical assistance; H. Yssel (INSERM U454, Montpellier, France) for the Ig secretion assay; D. Emilie (INSERM U131, Paris, France) for helpful discussions; and L. Leserman (Centre d’Immunologie Luminy, Marseille, France) and D. Blaise (Institut Paoli-Calmettes) for their critical reading of the manuscript.


    Abbreviations
 
ALL—acute lymphoblastic leukemia

APC—antigen-presenting cell

B-DC—B lymphocyte-derived DC-like cell

CLL—chronic lymphocytic leukemia

CTL—cytotoxic T lymphocyte

DC—dendritic cell

FISH—fluorescence in situ hybridization analysis

GM-CSF—granulocyte macrophage colony stimulating factor

HEV—high endothelial venule

LM—lymphoid malignancies

Mo-DC—monocyte-derived DC

PBMC—peripheral blood mononuclear cell

PCL—plasma cell leukemia

TNF—tumor necrosis factor


    References
 Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 

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