Phenotypic and functional characterization of monocyte-derived dendritic cells in chronic lymphocytic leukaemia patients: influence of neoplastic CD19+ cells in vivo and in vitro


Enrica Orsini MD, Dipartimento di Biotecnologie Cellulari ed Ematologia, University ‘‘La Sapienza’’, Via Benevento 6, 00161 Rome, Italy.


Dendritic cells (DCs) are the most potent antigen-presenting cells and are therefore an attractive option as antigen carriers in vaccination protocols. Chronic lymphocytic leukaemia (CLL) represents a potential good target for these approaches. The present study was designed to investigate the feasibility of generating in vitro fully functional DCs from peripheral blood (PB) monocytes of CLL patients at different phases of the disease. Although functional DCs could be obtained from CLL samples, in patients with active disease the expression of some co-stimulatory molecules appeared to be reduced. In contrast, DCs from CLL patients in remission showed no difference from those of normal controls. Moreover, patients with active disease produced DCs with reduced allostimulatory ability when compared with normal ones, whereas the functional capacities appeared to be restored in CLL DCs from remission patients. To more precisely assess the possible inhibitory effect of CLL cells on DC development, the influence of autologous leukaemic CD19+ cells on the generation of monocyte-derived CLL DCs in vitro was investigated. The addition of CLL neoplastic cells markedly affected monocyte-derived DC maturation. In conclusion, monocytes from CLL patients with active disease give rise to DCs, which show phenotypic and functional defects that are not observed in remission CLL patients. These results need to be taken into account in the design of DC-based immunotherapeutic approaches in CLL.

Chronic lymphocytic leukaemia (CLL), the most frequent leukaemia in the Western world (Catovsky & Foà, 1990), is a chronic disease characterized by the slow accumulation of mature B lymphocytes and by a relatively long median survival. No definitive curative strategy is currently available and the eradication of the disease is still an unattained goal (Rozman & Montserrat, 1995). It is a disease of the elderly, although about 20% of cases are less than 55 years of age at diagnosis (Mauro et al, 1999) and it is characterized by a heterogeneous clinical course. In recent years, it has been possible to associate the clinical heterogeneity with different biological markers – atypical morphology or immunophenotype, the presence of p53 abnormalities, the genetic profile and the presence of somatic mutations of the immunoglobulin (Ig) heavy chain variable genes – to an extent that some patients should not be treated, while others require a rapid and aggressive therapeutic intervention (Criel et al, 1997; Cordone et al, 1998; Damle et al, 1999; Dohner et al, 2000; Guarini et al, 2003).

In view of the nature of this incurable disease, in which a state of clinical remission without eradication can be obtained in many patients, minimal residual disease in CLL patients represents a rational setting for immunotherapeutic approaches. A large amount of immunological and clinical data is available on the potential role of anti-tumour vaccination in chronic lymphoproliferative diseases, particularly regarding the use of the idiotype (Id) of the tumour Ig (Ruffini et al, 2002; Rasmussen et al, 2003). Clinical responses and tumour regressions have been described in patients with B-cell lymphomas treated with vaccines containing the autologous leukaemic Id (Bendandi et al, 1999; Timmerman et al, 2002). The characteristic immunodepression associated with CLL, to such an extent that infectious complications are considered the main cause of mortality in these patients (Molica, 1994; Bartik et al, 1998), needs to be taken into careful account in the design of vaccination protocols. Multiple abnormalities have been documented over the years within the accessory non-leukaemic T-cell compartment of CLL patients, suggesting a chronic state of incomplete activation in vivo, with a consequent induction of an anergic state for CLL T cells (Semenzato et al, 1983; Foà, 1993; Prieto et al, 1993; Dianzani et al, 1994; Rossi et al, 1996). Moreover, functional defects have also been reported in the natural killer cell population (Foa et al, 1984, 1986) and, more recently, in the circulating dendritic cell (DC) compartment (Orsini et al, 2003). DCs have a pivotal role in the initiation and regulation of immune responses in vivo, and are responsible for the activation of naïve T cells and for the polarization of types 1 and 2 T-cell responses (Hart, 1997; Rissoan et al, 1999; Langenkamp et al, 2000; Liu, 2001). For these reasons, and because they are easily generated in vitro in large numbers from peripheral blood (PB) monocytes (Reid et al, 1992; Romani et al, 1994), DCs have been considered as ideal candidates for the design of anti-tumour vaccines, as potent adjuvant for the presentation of neoplastic antigens to the immune effectors. Some reports have recently described the possibility of obtaining functional in vitro monocyte-derived DCs from the PB of CLL patients (Rezvany et al, 2001; Vuillier et al, 2001). However, our group has described multiple defects within the in vivo circulating CLL DC compartment, which has proved to be a highly defective population, unable to adequately stimulate effector T cells, in accordance with the immunosuppression described in CLL patients (Orsini et al, 2003).

The present study was undertaken to obtain a more precise characterization of monocyte-derived DCs from CLL patients, in light of their possible clinical application as anti-leukaemic vaccines. The complex interconnections between the DC compartment and the neoplastic clone could help to explain the defects observed in vivo and also be useful in the design of DC-based anti-tumour vaccines.

Materials and methods

Patients and blood samples

We studied 10 CLL patients with active disease who had never received anti-leukaemic therapy and 10 patients in immunological remission (<5% CD5+/CD20+ double positive cells in the PB mononuclear population) after chemotherapy and rituximab (eight patients) or Campath-1H (two patients). The characteristics of the two groups of patients studied are summarized in Table I. Control samples were obtained from age-matched healthy donor buffy coats. PB mononuclear cells (PBMNCs) were separated by density gradient centrifugation (Lymphoprep; Nycomed Pharma, Oslo, Norway) and cultured in Roswell Park Memorial Institute 1640 medium (GIBCO, Life Technologies, Mulgrave, Vic., Australia) supplemented with 10% fetal bovine serum (FBS; HyClone, Logan, UT, USA), 0·3 mg/ml l-glutamine and 1% Pen-strepto (Euro-Clone, Milano, Italy), referred to as complete medium, at 37°C in 5% CO2 in air. In some cases, patient and control PBMNCs were cryopreserved in liquid nitrogen and thawed prior to use.

Table I.  Characteristics of chronic lymphocytic leukaemia patients.
PatientsSex (M/F)Age (years)*Leucocytes (×109/l)*% CD20/CD5+*Disease status
  1. SD, stable disease; PD, progressive disease; PR, partial remission; CR, complete remission.

  2. *Values are mean with range in parentheses.

Active disease (n = 10)7/362 (43–77)85·3 (17·8–138)78 (60–92)2 diagnosis
2 SD
6 PD
Remission (n = 10)8/257 (41–66)4·9 (1·8–9)1·8 (0–5)2 PR
8 CR

Generation of DCs from monocytes

Peripheral blood mononuclear cells from healthy donors and CLL patients, isolated as described above, were plated (1 × 107/3 ml per well) into six-well plates in complete medium. After 2 h of incubation at 37°C, non-adherent cells were removed by gentle washing and the adherent cells were cultured in complete medium supplemented with 100 ng/ml of interleukin (IL)-4 (R&D Systems, Abingdon, UK) and 100 ng/ml of granulocyte-macrophage colony-stimulating factor (GM-CSF) (R&D Systems). DCs were harvested for phenotypic or functional analysis after 7 d of culture. Samples from CLL patients with active disease were previously depleted of the overwhelming CD19+ B-cell population using immunomagnetic microbeads (Miltenyi Biotec, San Francisco, CA, USA). A mean of 7% CD19+ cells (range 3–12%) were detected in the samples after depletion, not significantly different from normal and CLL remission samples. CD19-depleted cells were processed for DC generation as described above.

The effects of CLL neoplastic cells on autologous DC maturation in vitro were examined by immunomagnetically sorting CD19+ cells from PBMNC samples of CLL patients with active disease (CD19 microbeads; Miltenyi Biotec). The remaining CD19 negative population was used to generate DCs as described, plating 5 × 106/3 ml per well, and 5 × 106 CD19+ cells/well were added after removal of the non-adherent fraction, together with IL-4 and GM-CSF. Wells without CD19+ neoplastic cells were used as controls.

To test the allostimulatory activity of monocyte-derived DCs, CD1a+ cells were sorted using immunomagnetic beads (Miltenyi Biotec) and used as stimulators in mixed leucocyte reaction (MLR) assays.

Cell surface FACS analysis

Cultured DCs were analysed by fluorescence multicolour flow cytometry. Cells were stained for 30 min at 4°C using Ca++/Mg++-free phosphate-buffered saline with 1% FBS as a diluent/wash buffer. Fluorescein isothiocyanate (FITC)-, phycoerythrin (PE)- and peridinin chlorophyll (PerCP)-conjugated monoclonal antibodies (mAbs) were used for triple-colour staining. Non-specific binding was measured using isotype-matched mAbs of irrelevant specificity. The antibodies used were purchased from Becton Dickinson (Mountain View, CA, USA) for isotype controls, PE-CD14, FITC-HLA-DR, PerCP-HLA-DR, PE-CD11c; Pharmingen (San Diego, CA, USA) for FITC- and PE-CD1a, FITC- and PE-CD83, FITC-CD80, PE-CD86, FITC-CD40; Dako (Glostrup, Denmark) for PE-HLA-ABC. Data acquisition was performed on a FACScan flow cytometer (FACScan; Becton Dickinson). For each sample, 10 000 events were evaluated. Mean fluorescence intensity (MFI) and the relative cell frequency expressing respective surface markers were analysed using the CellQuest software program (Becton Dickinson).

MLR assay

From 5 to 20 × 103 irradiated (30 Gy) CD1a+ DCs were used as stimulator cells for allogeneic PBMNCs obtained from healthy donors. Stimulators were added to the PBMNCs (105 cells/well) in 96-well round-bottomed microtest culture plates. After 5 d of incubation, cells were pulsed with 37 kBq 3H-TdR per well for the last 18 h, harvested and counted. Tests were performed in triplicate and results were expressed as mean counts per minute (c.p.m.). The levels of 3H-TdR uptake by stimulator cells alone were always less than 100 c.p.m.

Statistical analysis

The statistical significance between independent groups was determined using the Student's t-test for matched and unmatched pairs. P ≤ 0·05 was considered significant.


Monocyte-derived DCs from CLL patients with active disease express lower amounts of CD80 and CD40 co-stimulatory molecules

To assess the characteristics of monocyte-derived DCs in patients with CLL, DCs were generated in vitro from PB samples of 10 CLL patients with active disease by exposing adherence-isolated monocytes to GM-CSF and IL-4 for 7 d, as described in Materials and Methods. PB samples from 10 healthy donors were similarly processed and used as a control. Seven-day DCs were tested for different surface antigen expression, including CD1a, CD83, CD80, CD86, CD11c, CD40, HLA-I and HLA-II, and physical parameters. Table II shows the mean percentage of large (dendritic) cells in normal controls and in CLL DC cultures, and of positive cells for each surface antigen in the large cell population. The mean percentage of large DCs generated in healthy donor and CLL patient cultures was 48% and 26%, respectively, thus significantly reduced in CLL patients (P < 0·001). Moreover, the expression of both CD80 and CD40 was found to be significantly decreased in DCs from CLL patients compared with those generated from healthy donors: CD80 was expressed in 83% of gated large cells in normal DCs and in only 57% in CLL DCs (P < 0·001), CD40 expression was 90% and 62% respectively (P < 0·001). There were no significant differences in the expression of the remaining cell surface markers.

Table II.  Phenotype of monocyte-derived dendritic cells from normal donors and chronic lymphocytic leukaemia (CLL) patients.
% MarkerNormal donorsCLL patients with active diseaseCLL patients in remission
  1. Values are mean with range in parentheses.

  2. *P < 0·05 from normal donors.

  3. **P < 0·05 from CLL with active disease.

Large cells48 (28–64)26* (8–40)45** (26–77)
CD1a85 (59–97)72 (26–92)80 (50–89)
CD8631 (9–49)48 (14–75)41 (14–73)
CD8083 (57–98)57* (36–73)74** (51–86)
CD4090 (77–97)62* (37–82)89** (84–97)
CD83 7 (3–16)14 (2–25)10 (2–27)

In view of the design of possible vaccination protocols, it is conceivable that CLL patients with active disease do not represent an ideal population to study. Those patients that have already been treated, and are without clinical evidence of disease but not cured, should be considered as eligible candidates for immunotherapeutic approaches. We therefore investigated the phenotypic characteristics of monocyte-derived DCs obtained from 10 CLL patients in remission, defined as <5% of CD5+/CD20+ double positive cells in the PB. When compared with normal donors, CLL patients in remission showed no significant difference in the number of large DCs generated or in the expression of surface markers, including co-stimulation molecules (Table II). In contrast, the expression of both CD80 and CD40 was significantly higher than in DCs derived from CLL patients with active disease (P = 0·033 and P < 0·001 respectively).

Thus, monocyte-derived DCs from CLL patients with active disease showed phenotypic abnormalities that were no longer detectable after treatment in a phase of clinical remission. As an example of this evolution, Fig 1 shows the phenotypic profile of monocyte-derived DCs obtained from the same patient before and after treatment.

Figure 1.

Phenotype of monocyte-derived dendritic cells (DCs) before and after treatment. DCs were generated in vitro from peripheral blood (PB) monocytes of a chronic lymphocytic leukaemia (CLL) patient with active disease (panel A). Monocytes from the same patient were used for DC generation after obtaining clinical remission following therapy (panel B). The percentage of expression of surface antigens in the gated large cell population shown in the top left panel is indicated in the figure. Shaded histograms represent the isotype-matched negative controls.

Immunostimulatory activity of normal and CLL DCs

To assess the functional properties of monocytic DCs obtained from control donors and CLL patients, we tested their ability to stimulate the proliferation of allogeneic T cells. CD1a+ normal DCs, after 7 d of in vitro culture, demonstrated their functional maturity by exhibiting a good allostimulatory capacity in an MLR assay. Similar levels of activity were found in CD1a+ DCs obtained from CLL patients in clinical remission (Fig 2A). In contrast, a significantly reduced allostimulatory effect was shown by DCs obtained by CLL patients with active disease (Fig 2B).

Figure 2.

Allostimulatory ability of normal and chronic lymphocytic leukaemia (CLL) dendritic cells (DCs). CD1a+-sorted monocyte-derived DCs from healthy donors and CLL patients were irradiated and cultured with allogeneic peripheral blood mononuclear cells at different stimulator:responder ratio. 3H-TdR incorporation was measured after 5 d. Allogeneic mixed leucocyte reaction were performed with the same source of responder T cells for each experiment, involving one pair of normal/CLL DCs. Panel A: Normal DCs (solid lines) and CLL DCs from patients in remission (dotted lines): no differences are evident. Panel B: Normal DCs (solid lines) and CLL DCs from patients with active disease (dotted lines); CLL DCs show a statistically significant reduced allostimulatory activity (P = 0·009, 0·026 and 0·038 respectively). The assays were performed in triplicate and the mean values are shown. Triplicates were always consistent. c.p.m., counts per minute.

These results confirmed the altered functional properties of CLL DCs at diagnosis and the restoration of normal function after therapy.

Inhibitory effects of neoplastic CLL cells on autologous monocyte-derived DCs

As described, DCs generated from CLL patients with active disease presented phenotypic and functional defects. As we have previously found a profoundly defective circulating DC compartment in CLL patients and demonstrated that neoplastic CLL cells were capable of inhibiting the development of normal DCs (Orsini et al, 2003), we investigated whether the tumour clone could exert an inhibitory action on the in vitro development of monocyte-derived DCs from CLL patients. We used magnetic beads to separate CD19+ and CD19 cells from the PB of CLL patients with active disease. The adherent fraction from the CD19 population was divided between two wells and cultured with GM-CSF and IL-4 for 7 d to generate DCs. Leukaemic CD19+ cells were added at day 0 to one well to assess the effect on autologous DC development. As shown in Fig 3, DCs generated in the presence of autologous CLL neoplastic cells showed a markedly altered phenotype compared with the control cultures. Large DCs from the CD19+ well presented a markedly reduced expression of CD1a, CD80 and CD40. Furthermore, a large percentage of the DCs retained CD14 positivity, confirming an altogether less mature phenotype. Similar results were observed in three independent experiments. Thus, CD19+ tumour cells co-cultured with autologous DCs were shown to inhibit their development in vitro.

Figure 3.

Inhibitory effect of CD19+ cell on autologous dendritic cell (DC) development. Phenotype of chronic lymphocytic leukaemia (CLL) DCs obtained in vitro from the CD19 monocytic fraction of CLL patients with active disease (panel A). The addition of autologous CD19+ tumour cells to the cultures (panel B) induced a reduced expression of several surface antigens, including CD1a, CD80 and CD40. In contrast, CD14 expression increased under the influence of CD19+ cells. The percentage of expression of surface antigens in the gated large cell population shown in the top left panel is indicated in the figure. Shaded histograms represent the isotype-matched negative controls. Similar results were obtained in three different experiments.


The main objective of this study was to better characterize the phenotype and functions of monocyte-derived DCs from CLL patients in different phases of their disease and to try to define their interactions with the tumour clone. In a previously performed complete analysis of the circulating DC compartment in CLL patients with active disease, we have documented a complex array of defects in DC maturative potential and ability to activate T-cell effectors (Orsini et al, 2003). In particular, circulating CLL DCs appeared to show defective expression of the CD83 and CD80 (B7-1) antigens, but not of CD86 (B7-2), and also a reduced capacity to stimulate the proliferation of allogeneic T cells. However, the feasibility of generating in vitro functional DCs from PB monocytes in CLL patients with active disease has been reported, although the cultured CLL DCs were found to release an abnormal pattern of cytokines (Rezvany et al, 2001; Vuillier et al, 2001). As autologous DCs might represent a useful tool for immunotherapeutic approaches in patients with CLL and for the design of anti-tumour vaccines, we sought to better investigate which factors could impair DC function in vivo in CLL patients and how they could be overcome in vitro.

First, we examined the surface phenotype and allostimulatory ability of monocytic DCs from CLL patients with active disease. Although adequate numbers of morphologically normal DCs were obtained from all patients, it is of interest that a significant reduction in the expression of CD80 and CD40 co-stimulatory molecules was observed in comparison with normal controls. Expression of B7 molecules on the surface of antigen-presenting cells (APCs) is critical for T-cell activation, as T-cell receptor triggering in the absence of co-stimulation leads to T-cell anergy (Boussiotis et al, 1993). Although CD80 and CD86 are both activating partners for CD28 on T cells, they display different kinetics of expression and might also differ in their co-stimulatory properties. Indeed, it has been suggested that the interaction between CD28 and CD80 stimulates Th1 responses, while CD86 would stimulate Th2 responses (Bluestone, 1995; Freeman et al, 1995; Kuchroo et al, 1995; Thompson, 1995; Ranger et al, 1996). We found that circulating CLL DCs were defective in CD80 expression (Orsini et al, 2003) and the same held true in this study for monocyte-derived DCs from CLL patients, even if generated in the absence of neoplastic CD19+ B cells. Moreover, CD80 expression, and not CD86, was negatively affected by the addition of CLL cells in vitro, confirming the negative impact of these tumour cells on DC differentiation and function, as previously observed in vivo (Orsini et al, 2003). The different behaviour of the two co-stimulation antigens can thus be important in their functional consequences, possibly resulting in a different ability in directing T-cell differentiation. In this regard, the effects of CLL tumour cells seem to converge towards a decrease of CD80 expression, both in vivo and in vitro. A similar inhibiting action on type 1 T-cell responses might also be elicited by the decreased CD40 expression on CLL DCs, as CD40-mediated stimulation is known to induce DCs to release IL-12, the central cytokine that promotes Th1 differentiation and cell-mediated immune responses (Foy et al, 1996; Ford et al, 1999; Kikuchi et al, 2003).

Our results in CLL patients with active disease only partially agreed with previous studies that have reported a normal phenotype, when compared with healthy donors, of monocyte-derived CLL DCs. Vuillier et al (2001) did not find phenotypic and functional differences between DCs generated from five CLL patients and three normal donors. However, in addition to analysing a small number of patients, they did not investigate CD80 expression and CD40 expression was reported as MFI rather than as a percentage of positive cells. Rezvany et al (2001) reported a similar mean percentage of expression for CD80 and CD86 molecules (CD40 was not examined) between a group of five CLL patients and five control donors, but all their patients had non-progressive disease. Our population of patients was largely composed of patients with progressive disease that were analysed prior to the onset of therapy, and these clinical differences might have influenced DC generation and characteristics.

Next, we assessed if similar abnormalities, including the reduced ability to stimulate the proliferation of allogeneic T cells, were also present in CLL patients in remission after therapy. We found that neither the phenotypic characteristics nor the alloproliferative abilities of monocytic DCs from patients in remission were significantly different from those of normal donors. In particular, we were able to follow the increase in the expression of co-stimulatory molecules in DCs obtained from PB monocytes of one patient prior to therapy and after obtaining a complete clinical remission. These observations are of interest, as they confirmed the negative impact of the tumour clone on the in vitro-derived monocytic DCs, even in the absence of CD19+ neoplastic cells in the culture. However, the mechanisms responsible for this negative impact still need to be fully elucidated and are likely to be ascribed to defects in CLL circulating monocytes. The impaired release of cytokines (in particular tumour necrosis factor, interferon-α and IL-6) (Fernandez et al, 1986; Flieger et al, 1990; Dahlke et al, 1995; Anand et al, 1998), defects in the expression of leucocyte enzymes (normalized in remission of disease) (Zeya et al, 1979), inhibited chemotaxis (Norris et al, 1980) and reduced procoagulant activity (Cortellazzo et al, 1983) are all anomalies reported in the monocytic population of CLL patients with active disease. Moreover, complex effects of CLL B cells on the differentiation of CD14+ cells have recently been described by Tsukada et al (2002) who explored the role of monocyte-derived ‘nurse-like-cells’ on the survival of CLL neoplastic cells in vivo and in vitro. An impaired ability to give rise to phenotypically and functionally normal DCs in vitro could be part of this pattern of abnormal differentiation.

The possibility of generating normal monocytic DCs from CLL patients in remission is also important for the possible design of vaccination immunotherapeutic protocols, as this category of patients is the most likely to benefit from such approaches. Encouraging experimental and clinical results have been reported in different B-cell malignancies following vaccination with Id-specific vaccines (Bendandi et al, 1999; Timmerman et al, 2002) and in CLL with CD40L-transduced autologous leukaemic cells (Wierda et al, 2000). CLL may thus represent a good candidate for this type of alternative approach, given also the lack of eradicating therapies, and a number of preliminary in vitro studies have addressed this point. Goddard et al (2003) were successful in generating both human leucocyte antigen (HLA) class I- and HLA class II-restricted cytotoxic T-lymphocyte responses against autologous CLL B-cell targets using monocyte-derived DCs electrofused with CLL neoplastic cells. Autologous proliferative responses, but not cytotoxicity, were reported by Krackhardt et al (2002) using DCs pulsed with apoptotic bodies of CLL as APCs. The use of Id-pulsed DCs to generate a specific anti-tumour response has also been investigated, and the generation of CD8+ T-cell lines able to specifically recognize autologous tumour B cells has been described (Vuillier et al, 2003). However, these studies were all performed in patients with active disease. Taking into account the present data, which document phenotypic and functional differences between monocyte-derived DCs from CLL patients at diagnosis and after therapy, a deeper functional analysis of the ability of DCs from remission patients to induce protective autologous immune responses may be worthy. Indeed, our data suggest that DCs generated from patients in clinical remission after therapy might be superior in their allostimulatory activity compared with DCs from active disease CLL, indicating that patients in remission and with evidence of residual disease may be the category of CLL most suitable for vaccination strategies.

Further studies will be necessary to better clarify the functional characteristics of monocyte-derived DCs in CLL, especially in their relationship with effector T cells and in their ability to initiate protective autologous anti-tumour responses. In this study, we have shown that leukaemic CLL B cells have a negative impact on the generation of functional DCs, both in vivo (by altering the maturative capacity of circulating monocytes) and in vitro (by directly inhibiting the differentiation of functional DCs). In contrast, monocytic DCs generated from CLL patients in remission did not show differences compared with those obtained from normal donors. These findings should be taken into account when designing innovative immunotherapeutic strategies for the management of CLL patients.


This work was supported by Associazione Italiana per la Ricerca sul Cancro (AIRC), Milan; Istituto Superiore di Sanità, Rome; Ministero dell'Istruzione, Università e Ricerca (MIUR), Rome, Italy.