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Keywords:

  • dendritic cells;
  • CD14+ cells;
  • multiple myeloma;
  • cellular immunotherapy

Abstract

  1. Top of page
  2. Abstract
  3. Patients and methods
  4. Results
  5. Purification of circulating CD14+ cells by CliniMACS
  6. Phenotypic and functional characterization of CD14-derived DC
  7. Cryopreservation and thawing of preloaded DC, and functional studies
  8. Capability of IFN-α-2b (as an alternative to IL-4) to induce DC differentiation
  9. Discussion
  10. Acknowledgments
  11. References

Summary. Circulating monocytes from multiple myeloma patients enrolled in a clinical study of anti-idiotype vaccination were labelled with clinical-grade anti-CD14 microbeads and positively selected with the CliniMACS instrument. Cells were then grown, according to good manufacturing practice guidelines, in fetal-calf-serum-free medium in cell culture bags and differentiated to dendritic cells (DC) with granulocyte–macrophage colony stimulating factor plus interleukin 4 (IL-4), followed by either tumour necrosis factor-α (TNF-α) or a cocktail of IL-1β, IL-6, TNF-α and prostaglandin-E2. The CD14+ cell yield was increased from 17·6 ± 6·5% to 93·8 ± 6·3% (recovery 64·4 ± 15·4%, viability > 97%). After cell culture, phenotypic analysis showed that 86·7 ± 6·8% of the cells were DC: 2·27 ± 0·9 × 108 DC/leukapheresis were obtained, which represented 20·7 ± 4·6% of the initial number of CD14+ cells. Notably, the cytokine cocktail induced a significantly higher percentage and yield (28·6 ± 3% of initial CD14+ cells) of DC than TNF-α alone, with secretion of larger amounts of IL-12, potent stimulatory activity on allogeneic T cells and efficient presentation of tumour idiotype to autologous T cells. Storage in liquid nitrogen did not modify the phenotype or functional characteristics of preloaded DC. The recovery of thawed, viable DC was 78 ± 10%. Finally, interferon-α-2b was at least as efficient as IL-4 in inducing the differentiation of mature, functional DC from monocytes.

Dendritic cells (DC) are professional antigen-presenting cells (APC) specialized in capturing antigens (Ags) and transforming them in to peptide fragments that bind to major histocompatiblity complex (MHC) molecules (Bancherau & Steinman, 1998). DC are the most potent stimulators of T cell-mediated immune responses and they are unique in that they stimulate not only memory but also naive T cells. A growing body of evidence supports the role of DC in antitumour immunity (Nestle et al, 1998; Thurner et al, 1999a; Brossart et al, 2000; Kugler et al, 2000; Fong et al, 2001). Recent data on DC vaccination of multiple myeloma (MM) and lymphoma patients indicated that stimulation of an anti-idiotype (Id) T-cell response may sometimes be feasible (Hsu et al, 1996; Reichardt et al, 1999; Timmerman et al, 2002). The development of simple methods for ex vivo production of large numbers of clinical-grade DC from haematopoietic precursors is a prerequisite for a widespread clinical application of DC-based immunotherapy. However, current protocols to generate human DC often exert insufficient maturation stimuli and rely on clinically inappropriate techniques, such as isolation of monocytes (Mo) by non-standardized plastic adherence and use of fetal calf serum (FCS) or (easily contaminated) tissue culture flasks.

We recently showed that peripheral blood (PB) Mo from MM patients can be induced to differentiate into fully functional, mature CD83+ DC (Ratta et al, 2000). Following immunomagnetic adsorption of CD14+ cells, the Mo were stimulated in serum-replete cultures by a combination of granulocyte–macrophage colony-stimulating factor (GM-CSF) and interleukin 4 (IL-4), with sequential use of tumour necrosis factor-α (TNF-α) as a maturation factor. The resulting CD83+ DC-enriched cell population was highly efficient in priming allogeneic and autologous T lymphocytes in response to the patient-specific tumour Id.

We have now scaled up our manufacturing protocol for application in a phase I–II clinical trial of anti-Id vaccination with DC in MM patients. To this end, we have adapted our original method to allow reproducible generation of mature and functional myeloid DC from unprimed leukapheresis products in accordance with Good Manufacturing Practice (GMP) guidelines.

Leukocyte aphereses.  PB cells were obtained from 16 pretreated MM patients in steady-state conditions (i.e. no mobilizing treatments were administered). All patients had stage III disease according to Durie–Salmon classification and had been previously submitted to one (= 10) or two (= 6) courses of myeloablative treatment supported by reinfusion of autologous stem cells. At the time of study, 15/16 patients showed responsive disease (i.e. > 50% reduction of tumour burden). Four patients were entered in a trial of preclinical evaluation of large-scale positive selection of CD14+ cells (numbers 9–12; Table I). Subsequently, eight additional patients (numbers 1–8; Table I) were enrolled in a phase I–II clinical trial of anti-Id vaccination. Concomitantly, four more patients had their PB CD14+ cells purified and split in to two aliquots to compare different culture conditions [i.e. IL-4 versus interferon (IFN)-α-2b to generate DC; TNF-α versus the cytokine combination for DC maturation, see below]. Two blood volumes leukaphereses were performed using either a Fenwal CS3000 continuous-flow blood cell separator (Baxter Healthcare, Deerfield, IL, USA) or a Cobe Spectra separator (Cobe BCT, Lakewood, CO, USA), as previously reported (Lemoli et al, 1997). The protocol was approved by the Ethical Committee of the University Hospital and all the subjects gave written informed consent.

Table I.  Positive selection of CD14+ monocytes by CliniMACS.
Patient numberCD14+ × 109 baseline% CD14+ baselineCD14+ × 109 after selectionPurity percentageRecovery percentageViability percentage
11·0812·70·8598·378·7 96
22·9114·51·8195·762·2100
31·53180·8595·955·6100
42·3919299·283·7 96
51·2914·11·2398·895·3 97
62·1813·61·2095·855 90
71·7912·21·1296·962·6 96
82·8414·21·9891·269·7 99
93·30221·298539 90
102·20181·169052·7 85
110·9317·30·507953·8 80
121·4335·90·939165 90
Mean ± SD1·99 ± 0·7717·6 ± 6·51·24 ± 0·4793·8 ± 6·364·4 ± 15·4 97·2 ± 2·9

Clinical-grade purification of CD14+ cells.  Isolation of CD14+ monocytes was performed by immunomagnetic labelling of target cells followed by an automated separation process using the CliniMACS device (Miltenyi Biotec, Bergisch Gladbach, Germany), according to the manufacturer's instructions. Briefly, up to 2 × 1010 PB mononuclear cells (MNC) were washed with phosphate-buffered saline (PBS) and 1% human albumin to reduce the labelling volume to 50 ml. The cells were then incubated with a clinical-grade anti-CD14 monoclonal antibody (mAb) conjugated to MicroBeads (CliniMACS CD14 Reagent; Miltenyi Biotec) for 15 min on a rotating plate. Removal of excess reagent was performed by two washes using a Cobe 2991 cell processor (Gambro BCT, Stockholm, Sweden). CD14+ cells were subsequently purified on CliniMACS by using the ‘Enrichment 1·1’ program. Positively selected CD14+ cells were counted, analysed for cell viability and phenotype, and used to generate DC. Assessment of CD14+ cells before and after the separation process was carried out by labelling the target cells with anti-CD14 phycoerythrin (PE) mAb (Becton Dickinson, San Jose, CA, USA), followed by flow cytometric re-analysis of purified cell fractions performed on a gated population set on scatter properties, using the FACScan equipment (Becton Dickinson) (Ratta et al, 2000, 2002). A minimum of 10 000 events was collected in list mode using facscan software.

Generation of mature DC in liquid culture.  DC were generated from PB CD14+ cells based on previously described methods (Ratta et al, 2000; Ratta et al, 2002), incorporating important modifications. In particular, 2 × 106 purified CD14+ cells were cultured in 1 ml of serum-free medium (CellGro DC medium; CellGenix, Freiburg, Germany) supplemented with 1% serum from AB normal donors and 50 ng/ml of GM-CSF (Myelogen, Schering Plough, Kenilworth, NJ, USA) and 800 IU/ml of IL-4 (CellGenix). In four experiments (as shown), IL-4 was replaced by 500, 1000 or 2000 IU/ml of IFN-α-2b (Intron-A; Schering Plough). After 6 d of culture, 25 ng/ml of TNF-α (CellGenix) were added to the culture for 36 h to induce terminal maturation of DC (Ratta et al, 2000). In selected experiments (n = 4), TNF-α (10 ng/ml) was used as a maturation stimulus in addition to IL-1β (10 ng/ml), IL-6 (1000 IU/ml) and prostaglandin E2 (PGE2; 1 μg/ml) for 36 h. All these cytokines (purchased from CellGenix) were manufactured in accordance with GMP guidelines. Pulsing with patient-specific tumour Id (IgG; 50 μg/ml), as whole protein, was performed before the maturation of DC, as previously reported (Ratta et al, 2000). In some cases, DC were co-incubated with 50 μg/ml of Id-specific peptides (unpublished observations). Keyhole limpet haemocyanin (KLH; 50 μg/ml) was always added to tumour Id as a helper epitope to improve the potency of the vaccine (Timmerman & Levy, 2000). Cultures were maintained at 37°C in 5% CO2 in cell culture bags (SteriCell; Nexell Therapeutics, Rome, Italy) by replacing culture medium and cytokines at d +3. Generation of functionally active DC was assessed by phase contrast microscopy, immunophenotyping, evaluation of IL-12 secretion and mixed lymphocyte reactions (MLR) (see below).

Cryopreservation and thawing of DC.  Mature DC were adjusted to a concentration of 1 × 107 cells/ml, cryopreserved at −1°C/min in 10% dimethylsulphoxide (DMSO) and 20% AB plasma using a controlled-rate freezing method, and stored at −196°C in liquid nitrogen as previously reported (Lemoli et al, 1996). Frozen volumes did not exceed 4·5 ml. Frozen cells were thawed rapidly in a 40°C water bath, centrifuged once to remove DMSO and assayed as described above. Viability was determined upon thawing by the trypan blue exclusion assay.

Immunophenotype studies.  Dual-colour immunofluorescence was performed using the following panel of mAbs: PE-or fluorescein isothiocyanate (FITC)-conjugated anti-human CD1a (Pharmingen, San Diego, CA, USA); FITC-anti-human CD86 (Pharmingen); FITC-BB1/B7 (anti-CD 80; Becton Dickinson); FITC-anti-human HLA-DR (Becton Dickinson); FITC- or PE-Leu-M3 (anti-CD 14; Becton Dickinson); FITC-Leu-4 (anti-CD3; Becton Dickinson); PE-anti-human CD83 (Immunotech, Marseille, France); FITC-anti-human CD40 (Pharmingen). Negative controls were isotype-matched irrelevant mAbs (Pharmingen and Becton Dickinson). Cells were incubated in the dark for 30 min at 4°C in PBS and 1% bovine serum albumin (BSA). After washing, cells were resuspended in PBS and 1% paraformaldehyde and analysed as described above.

FITC-dextran assay.  To evaluate the capacity of DC to uptake soluble antigens from the culture medium, DC were incubated with 1 mg/ml of FITC-dextran at 37°C or at 0°C for 1 h. Uptake was stopped by adding ice-cold PBS followed by four washes in a refrigerated centrifuge (Ratta et al, 2000). Cells were then analysed by flow cytometry using a FACScan (Becton Dickinson).

Assessment of IL-12 production. Mature DC (106/ml) were assayed in duplicate for IL-12 production in serial dilutions, using an enzyme-linked immunosorbent assay kit that evaluated the biologically active p75 heterodimer (R & D Systems, Minneapolis, MN, USA).

Purification of tumour Id.  Monoclonal IgG of MM patients were purified from serum using protein-G columns (Hi Trap protein G Sepharose; Pharmacia Biotech, Uppsala, Sweden), as reported elsewhere (Goding, 1996). The purity of the M component, evaluated by sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE; Pharmacia Biotech), was always greater than 90%.

Activation of allogeneic and autologous T-cell proliferation.  To test their allogeneic stimulatory activity, fresh and cryopreserved DC were irradiated (3000 cGy) and tested as stimulators in primary MLR (Ratta et al, 2000, 2002). Cells were resuspended in Roswell Park Memorial Institute (RPMI)-1640 medium, 25 mmol/l HEPES, antibiotics and 15% AB human serum that had been inactivated at 56°C for 30 min. allogeneic PB MNC (5 × 104) were mixed with decreasing numbers of stimulators in round-bottomed 96-well plates for 6 d at 37°C in a 5% CO2 humidified atmosphere. Cells were pulsed with 37 kBq/well 3H-thymidine for 18 h before harvest on d 6. The stimulation index (SI) was calculated for each individual experiment as follows: SI = counts per minute (c.p.m) of T-cell responders plus stimulators, divided by c.p.m. of T-cell responders. Autologous MLRs were set up to demonstrate the capacity of cultured DC to process and present the patient-specific Id to T cells (Ratta et al, 2000, 2002). Briefly, 105 PB T cells were co-incubated with a fixed number of autologous APC (n = 3000) with or without 50 μg/ml of purified Id from MM patients. T-cell proliferation was measured as follows: SI = c.p.m. of T-cell responders plus Ag-pulsed stimulators, divided by c.p.m. of T-cell responders plus stimulators.

Statistical analysis.  Results are expressed as mean ± standard deviation (SD). Where indicated, analysis was performed by means of the non-parametric paired Wilcoxon rank-sum test.

Purification of circulating CD14+ cells by CliniMACS

  1. Top of page
  2. Abstract
  3. Patients and methods
  4. Results
  5. Purification of circulating CD14+ cells by CliniMACS
  6. Phenotypic and functional characterization of CD14-derived DC
  7. Cryopreservation and thawing of preloaded DC, and functional studies
  8. Capability of IFN-α-2b (as an alternative to IL-4) to induce DC differentiation
  9. Discussion
  10. Acknowledgments
  11. References

In the first set of experiments, CD14+ cells were highly purified from unprimed leukaphereses of 12 MM patients, using the CliniMACS high-gradient magnetic separation column. Four patients (numbers 9–12; Table I) entered in a trial of preclinical evaluation of large-scale positive selection of CD14+ cells whereas eight additional patients (numbers 1–8; Table I) were enrolled in a phase I–II clinical trial of anti-Id vaccination. The mean number of nucleated cells in the leukapheresis products was 13·9 ± 5·5 × 109, of which 17·6 ± 6·5% were CD14+ (Table I). After purification, we recovered 1·3 ± 0·48 × 109 nucleated cells, of which 93·8 ± 6·3% were CD14+. The mean overall recovery of CD14+ elements was 64·4 ± 15·4% and cell viability was greater than 97% (Table I). A representative example of enrichment of CD14+ cells is shown in Fig 1. The CD14 cells were mainly T lymphocytes (always > 70%), which were cryopreserved and used as responders in autologous MLR.

image

Figure 1. Clinical-grade purification of PB CD14+ cells. Leukapheresis collections and enriched populations were double stained with anti-CD14 and anti-CD3 mAbs. The percentages of CD3+ and CD14+ cells are shown in the diagrams. The results are representative of 16 consecutive procedures.

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Maturation using TNF-α alone.  Using cell culture bags, a mean of 1·16 × 109 highly purified CD14+ cells (Table II) were cultured in FCS-free medium supplemented with 1% AB human serum and were then differentiated into immature DC by treatment with GM-CSF and IL-4, as previously reported (Ratta et al, 2000) (Table II). After 6 d, TNF-α was added to the culture for 36 h as a maturation stimulus. Based on our previous finding (Ratta et al, 2000) that the optimal timing for pulsing DC with soluble Ags is during maturation, when the highest capacity of Ag uptake and processing occurs, tumour Id and/or KLH were added to the culture 2 h before addition of TNF-α. At the end of culture, we obtained 2·27 ± 0·9 × 108 DC/leukapheresis, which represented 20·7 ± 4·6% of the initial number of CD14+ cells (Table II). Phenotypic analysis (Table II) showed that 86·7 ± 6·8% of DC expressed HLA-DR, CD40 and co-stimulatory molecules, whereas CD14 and CD1a were (as expected) downregulated. In this set of experiments, the percentage of fully mature CD83+ cells produced was 28·2 ± 18·1% of DC (see below).

Table II.  Generation of DC from highly purified CD14+ cells.
Patient numberCD14+ cultured × 109Tumour antigenDC generated × 106DC purity* (%)Yield of DC (%)
  1. *Co-expression of CD 40+ and HLA DR+ antigens and expression of co-stimulatory molecules.

10·85Idiotype + KLH169·196·119·9
21·81Idiotype + KLH365·68820·2
30·85Peptides + KLH196·396·823·1
42Idiotype + KLH36083·218
51·23Peptides + KLH276·78522·5
61·20Peptides + KLH304·895·425·4
71·12Idiotype + KLH26190·323·3
81·98Peptides + KLH209·98410·6
90·80KLH1087713·5
101·12KLH244·28121·8
110·50KLH121·58624·3
120·46KLH116·87825·4
Mean ± SD1·16 ± 0·53 227·8 ± 9086·7 ± 6·820·7 ± 4·6

Maturation using TNF-α, IL-1β, IL-6 and PGE2.  Based on the previous results, we reasoned that TNF-α may be an insufficient stimulus to drive DC maturation in FCS-free medium. Thus, with four additional patients, immature DC (i.e. DC recovered after 6 d of treatment with GM-CSF/IL-4) were incubated using a cocktail of cytokines and prostaglandins, comprising TNF-α, IL-1β, IL-6 and PGE2, which has been shown to induce efficient maturation of Mo DC in preclinical studies (Jonuleit et al, 1997; Thurner et al, 1999b). In this set of experiments, both CD83+ and co-stimulatory molecules were significantly upregulated by the cytokines cocktail (Fig 2). With respect to TNF-α alone, this combination induced higher percentages of CD83+ (65·2 ± 10·3%vs 28·2 ± 18·1%; P < 0·02) and CD80+ (64·6 ± 9%vs 47·1 ± 15·8%; P < 0·05) cells and a lower percentage of CD1a+ DC (1·9 ± 1%vs 12·1 ± 8·3%; P < 0·01) (Fig 3). The higher degree of maturation induced by the cocktail of cytokines was also demonstrated by the decreased capacity of DC to uptake FITC-dextran as compared with TNF-α-matured DC (Fig 4). These data translate into a higher yield of viable DC (28·6 ± 3% of the initial CD14+ cells) with a purity of 94·3 ± 7%. Moreover, DC matured by TNF-α, IL-1β, IL-6 and PGE2 secreted larger amounts of IL-12 (4·5 ± 1 ng/ml vs 1·5 ± 0·7 ng/ml; P = 0·03).

image

Figure 2. The phenotypic profile of DC generated ex vivo from highly purified CD14+ cells in the presence of GM-CSF and IL-4 for 6 d (immature DC) or matured with the combination of TNF-α, IL-1, IL-6 and PGE2 (mature DC). Overlay diagrams show the expression of the relevant Ags versus isotype-matched Ab (negative controls). The figure shows both the percentage of positive cells and the mean fluorescence intensity (MFI) value. The MFI indicates the upregulation of CD83, HLA-DR and co-stimulatory molecules, and down regulation of CD1a. The results are representative of four different experiments. See the text for further details.

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image

Figure 3. Phenotypic analysis of Mo DC generated ex vivo in presence of GM-CSF, IL-4 and matured with either TNF-α (TNF-α) alone or the cocktail of TNF-α, IL-1, IL-6 and PGE2 (PGE2). The combination of cytokines induced a significantly higher percentage of CD83+, CD80+ cells and a lower percentage of CD1a+ cells. *P-values < 0·05. The results are expressed as the mean ± SD of four different experiments.

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image

Figure 4. Antigen uptake capacity (FITC-dextran) of immature DC and DC matured after incubation with TNF-α alone or a combination of TNF-α, IL-1, IL-6 and PGE2. The representative light scatter profile of the cells is also shown. Overlay diagrams show the expression of FITC-dextran versus the negative control. In this representative experiment, the percentage of FITC-dextran-positive cells decreased from 85% to 35% when TNF-α was used alone and down to 4% when the cytokine cocktail was tested.

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Cryopreservation and thawing of preloaded DC, and functional studies

  1. Top of page
  2. Abstract
  3. Patients and methods
  4. Results
  5. Purification of circulating CD14+ cells by CliniMACS
  6. Phenotypic and functional characterization of CD14-derived DC
  7. Cryopreservation and thawing of preloaded DC, and functional studies
  8. Capability of IFN-α-2b (as an alternative to IL-4) to induce DC differentiation
  9. Discussion
  10. Acknowledgments
  11. References

Mature, preloaded DC were stored in liquid nitrogen for periods of time ranging from 7 to 90 d. Upon thawing, 78 ± 10% of the cells were viable regardless of the duration of cryopreservation and the type of maturation stimulus. The phenotype of DC was not altered by cryopreservation (Fig 5). The immunostimulatory activity of fresh and cryopreserved Mo DC was also determined by a one-way MLR assay. As shown in Fig 6A, the alloreactivity of the DC was not significantly affected by freezing/thawing procedures.

image

Figure 5. Phenotypic profile of DC analysed before and after cryopreservation in liquid nitrogen. The results are expressed as the mean ± SD of eight different experiments.

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image

Figure 6. T-cell stimulatory capacity of fresh and cryopreserved, clinical-grade, CD14-derived DC. Increasing numbers of DC were used to stimulate 5 × 104 allogeneic cryopreserved MNC (A); autologous MNC were used as negative control. The data reported are the mean ± SD of 10 different experiments performed in triplicate. (B) T cells (1 × 105) were incubated with 3000 fresh and cryopreserved DC preloaded with the tumour Id. Autologous DC alone (which represented the negative control; stimulation index = 1) gave 5300 ± 1880 c.p.m. The data reported are the mean ± SD of nine different experiments performed in triplicate.

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To further characterize the functional capacity of Mo DC derived from MM patients, we compared the ability of cryopreserved DC to present the patient-specific Id to autologous T cells with that of DC tested before freezing. Notably, DC were loaded with tumour Id before cryopreservation. As shown in Fig 6B, both fresh and frozen Mo DC were capable of inducing proliferation of autologous T cells in response to the tumour Ag. Taken together, these results demonstrated that the DC did not lose their functional capacity following cryopreservation. In particular, they could be effectively loaded with the tumour Id (either as whole protein or as Id-derived peptides) before storage.

Capability of IFN-α-2b (as an alternative to IL-4) to induce DC differentiation

  1. Top of page
  2. Abstract
  3. Patients and methods
  4. Results
  5. Purification of circulating CD14+ cells by CliniMACS
  6. Phenotypic and functional characterization of CD14-derived DC
  7. Cryopreservation and thawing of preloaded DC, and functional studies
  8. Capability of IFN-α-2b (as an alternative to IL-4) to induce DC differentiation
  9. Discussion
  10. Acknowledgments
  11. References

In a final set of experiments, we investigated whether IL-4 could be replaced, in a clinical setting, by IFN-α-2b to drive the differentiation of DC from CD14+ cells. First, we verified whether incubation of highly purified Mo with IFN-α-2b and GM-CSF in FCS-free culture medium led to the differentiation of DC (Santini et al, 1999; Parlato et al, 2001). In these small scale preliminary experiments, IFN-α-2b was used at three different doses (500, 1000 and 2000 U/ml) and the duration of culture varied from 3 to 6 d. In line with previous reports (Santini et al, 1999; Parlato et al, 2001), we found that 1000 U/ml of IFN-α-2b was the optimal dose for development of fully functional DC. However, unlike others (Santini et al, 1999; Parlato et al, 2001), we found that 6 d of incubation were needed for the production of DC in FCS-free medium. Moreover, incubation with IFN-α-2b and GM-CSF alone (i.e. without maturation stimuli) induced immature DC that retained a high capacity of protein uptake (86% of the cells were positive for FITC-dextran) with a low percentage of CD83+ cells (< 20%). Thus, in the following clinical scale experiments, CD14+ cells were enriched from the leukapheresis products of four MM patients. Purified monocytes were split in to two aliquots, and grown in the presence of GM-CSF and either IL-4 or IFN-α-2b (1000 U/ml) for 6 d. The maturation phase was carried out by adding TNF-α, IL-1β, IL-6 and PGE2. The yield of DC was similar after incubation with IFN-α-2b or IL-4 [28 ± 6%vs 24·5 ± 4% of the initial number of CD14+ cells; P = not significant (NS)]. Figure 7 shows a comparison of the representative phenotypic profiles obtained after maturation. Notably, DC treated with IFN-α-2b showed a remarkably high expression of HLA-DR, CD40, co-stimulatory molecules and CD83 after maturation. Moreover, DC treated with 1000 U/ml of IFN-α-2b showed a potent allostimulatory activity, equivalent to that of IL-4-derived cultures (Fig 8). When we assessed IL-12 secretion, we found no difference between IL-4- or IFN-derived DC: 2·8 ± 0·8 ng/ml vs 2·4 ± 0·4 ng/ml (P = NS). Taken together, these comparative experiments demonstrated that IFN-α was at least as efficient as IL-4 in inducing differentiation of Mo to the DC lineage.

image

Figure 7. Phenotypic characterization of DC generated ex vivo from purified CD14+ cells in the presence of GM-CSF and either IL-4 or IFN-α-2b and matured with TNF-α, IL-1, IL-6 and PGE2. The overlay diagrams show the expression of the relevant antigens on positive cells with respect to isotype-matched Ab (negative controls). Of note, the MFI values of HLA-DR (1420 vs 569), CD40 (175 vs 112), CD80 (185 vs 112) and CD86 (290 vs 162) were higher in IFN-treated DC as compared with IL-4-treated DC. The results are representative of four different experiments.

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Figure 8. MLR stimulatory capacity of irradiated Mo DC generated ex vivo in the presence of GM-CSF and either IL-4 or IFN-α-2b (1000 and 2000 U/ml) and matured with TNF-α, IL-1, IL-6 and PGE2. When IFN-α-2b was used at the concentration of 500 U/ml, the results were superimposable with those observed with 2000 U/ml (data not shown). The results are representative of four different experiments performed in triplicate.

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Discussion

  1. Top of page
  2. Abstract
  3. Patients and methods
  4. Results
  5. Purification of circulating CD14+ cells by CliniMACS
  6. Phenotypic and functional characterization of CD14-derived DC
  7. Cryopreservation and thawing of preloaded DC, and functional studies
  8. Capability of IFN-α-2b (as an alternative to IL-4) to induce DC differentiation
  9. Discussion
  10. Acknowledgments
  11. References

We and others have previously shown the feasibility of generating large numbers of DC from steady-state or mobilized MNC from MM patients (Pfeiffer et al, 1997; Wen et al, 1998; Raje et al, 1999; Ratta et al, 2000; Tarte et al, 2000). However, whereas the majority of the current protocols for the generation of Mo DC involve the use of multistep Percoll gradients followed by further depletion of T and B cells or isolation of monocytes by plastic adherence, we applied (Ratta et al, 2000) a simple and highly reproducible protocol for the positive selection of CD14+ monocytes. Stimulation of CD14+ cells by the sequential combination of GM-CSF, IL-4 and TNF-α produced a generation of DC that were highly efficient in stimulating allogeneic T cells and autologous T lymphocytes in response to the tumour Ag.

In the present study, we scaled up our original method (suitably modified to fulfil GMP conditions) to allow its use in a phase I–II clinical trial of anti-Id vaccination with DC in MM patients.

First, we took advantage of a licensed GMP technology for positive selection of CD14+ cells (i.e. DC precursors). The use of clinical-grade anti-CD14 labelling reagent and CliniMACS enabled the reproducible large-scale purification of circulating Mo. The overall efficiency did not differ from that obtained experimentally in vitro (Ratta et al, 2000). Similar data have recently been briefly reported in the context of a preliminary study on enrichment of PB CD14+ cells from chronic myelogenous leukaemia patients (Padley et al, 2001). However, the authors did not report either a full phenotypic profile of the DC or functional assays to demonstrate the efficiency with which they could stimulate T cells. In the present study, we were able to show that CD14-derived DC are fully functional and capable of inducing T-cell proliferation to the patient-specific tumour Id. Therefore, thanks to the ease and reproducibility of positive selection of CD14+ cells, we believe that this approach could significantly help to improve the feasibility of clinical trials of DC-based immunotherapy. In fact, whereas Mo DC have been shown by us and others to be efficient in tumour Ag presentation, circulating DC isolated from MM patients have an impaired capacity of presenting the patient-specific Id to T cells (Ratta et al, 2002), and CD34+ cells, another tested source of DC (Titzer et al, 2000), have a limited proliferative potential and provide a lower yield (Ratta et al, 2000).

In this trial, we performed DC cultures in cell culture bags (a closed system that minimizes adventitial micro-organism contamination) in the absence of FCS, which is strictly contra-indicated for clinical use. However, we found that, under these conditions, TNF-α alone, which was used in the first eight patients, was an insufficient maturation stimulus for DC. To provide a viable alternative, we confirmed, in a clinical trial, that incubation with a cocktail of pro-inflammatory cytokines, including TNF-α, IL-1β, IL-6 and PGE2, results in the efficient maturation of Mo DC and higher production of IL-12 (Jonuleit et al, 1997; Thurner et al, 1999b). This finding is particularly important clinically, as only fully mature CD83+ DC remain unaffected by inhibitory factors, such as IL-10 and vascular endothelial growth factor (Gabrilovich et al, 1996; Steinbrink et al, 1997), and induce Th1 polarization and cytotoxic T-cell response (Bhardwaj et al, 1994; Macatonia et al, 1995). In this regard, it will be interesting to see whether the MM patients that enter our phase I–II clinical trial and are reinfused with a higher percentage of CD83+ cells will develop a more effective anti-Id T-cell response, in vivo, compared with patients receiving TNF-α-matured DC.

We chose to select CD14+ cells from unprimed PB for two reasons. First, preliminary experiments showed that after G-CSF mobilization a large number of mature myeloid CD14+/CD15+ cells co-purify with monocytes. These cells readily underwent apoptosis in culture, thus significantly affecting the yield and function of DC (data not shown). In addition, recent data demonstrated that G-CSF-treated monocytes are not an efficient source of fully functional DC (Volpi et al, 2001). Mature and loaded DC were cryopreserved and, upon thawing, proved to be equivalent to fresh DC with respect to both phenotype and T-cell stimulatory capacity. Thus, we were able to avoid the repetitive production of DC before each vaccination. This approach also simplified the execution of safety and quality tests, which would otherwise have had to be performed on several cellular products.

The availability of cryopreserved preloaded DC also greatly facilitates the clinical use of DC. In fact, in our phase I–II trial of anti-Id DC vaccination, cells from a single apheresis are planned to be stored in the gas phase of liquid nitrogen and subsequently used for three subcutaneous and two intravenous administrations, 2 weeks apart, at cellular doses of 5 × 106/1 × 107/5 × 107 and 1 × 107/5 × 107 respectively. The overall requirement of DC for the entire program is 1·25 × 108, well below the mean number of thawed and viable DC produced for each patient. However, although our protocol provides > 2·2 × 108 well-characterized DC/steady-state leukapheresis, the yield of DC was rather low (approximately 30% of the initial number of monocytes) and requires further improvement.

In the last set of experiments, we investigated whether IFN-α-2b, in a clinical setting, could replace IL-4 for the differentiation of DC from CD14+ cells. IL-4 is no longer produced for clinical trials and, although it is GMP-manufactured by a few companies, the costs are exceedingly high (in the range of $8–9000 per patient). IFN-α-2b has been shown to induce rapid (3 d) differentiation of GM-CSF-treated monocytes into DC that exert a potent APC activity in vitro and in vivo. In our study, IFN-α-2b gave results comparable to (or marginally better than) those of IL-4 in terms of DC phenotype, IL-12 production and alloreactivity. In addition, despite the need to extend the culture time to 6 d under FCS-free conditions, we calculated that the cost of IFN-α-2b treatment would be as low as $90 per patient. Further experiments are underway to evaluate whether DC generated in presence of IFN-α-2b are capable of presenting tumour Id to autologous T cells.

In summary, our data demonstrated that: (1) the availability of an anti-CD14 labelling reagent, manufactured in compliance with GMP guidelines, enabled the large-scale purification of circulating Mo suitable for clinical trials; (2) a 2-d incubation in the presence of TNF-α, IL-1β, IL-6 and PGE2 enabled the efficient upregulation of CD83 on DC and greater secretion of IL-12; (3) cryopreservation did not affect the phenotype and functional characteristics of preloaded Mo-derived DC; (4) IFN-α-2b may be used instead of IL-4 (which may be no longer available for clinical use) for differentiation of CD14+ cells to the DC lineage.

Therefore, the present work shows how our original approach for the generation of DC can be readily scaled up to a clinical level. We hope that the optimization of strategies to produce fully functional DC may induce Id-specific immune responses in a larger cohort of MM patients (Reichardt et al, 1999).

Acknowledgments

  1. Top of page
  2. Abstract
  3. Patients and methods
  4. Results
  5. Purification of circulating CD14+ cells by CliniMACS
  6. Phenotypic and functional characterization of CD14-derived DC
  7. Cryopreservation and thawing of preloaded DC, and functional studies
  8. Capability of IFN-α-2b (as an alternative to IL-4) to induce DC differentiation
  9. Discussion
  10. Acknowledgments
  11. References

The work was supported by: Associazione Italiana per la Ricerca sul Cancro (AIRC), Milan, Italy; CNR-MIUR Rome, Italy; ‘Progetto Finalizzato Oncologia’, University of Bologna (fondi ex 60%). The authors wish to thank Mr Robin M. T. Cooke for scientific editing.

References

  1. Top of page
  2. Abstract
  3. Patients and methods
  4. Results
  5. Purification of circulating CD14+ cells by CliniMACS
  6. Phenotypic and functional characterization of CD14-derived DC
  7. Cryopreservation and thawing of preloaded DC, and functional studies
  8. Capability of IFN-α-2b (as an alternative to IL-4) to induce DC differentiation
  9. Discussion
  10. Acknowledgments
  11. References
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