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Monitoring and isolation of blood dendritic cells from apheresis products in healthy individuals: a platform for cancer immunotherapy
Journal of Immunological Methods 267 (2002) 199 – 212 www.elsevier.com/locate/jim
Monitoring and isolation of blood dendritic cells from apheresis products in healthy individuals: a platform for cancer immunotherapy J. Alejandro Lo´pez a, Georgina Crosbie a, Cathryn Kelly b, Ann Marie McGee a, Katrina Williams b, Slavica Vuckovic a, Robert Schuyler c, Robyn Rodwell b, Sue J. Wright b, Kerry Taylor b, Derek N.J. Hart a,* a
Mater Medical Research Institute, Aubigny Place, South Brisbane 4101, Australia b Mater Adult Hospital, Raymond Terrace, South Brisbane 4101, Australia c Gambro BCT, Lakewood, CO 80215, USA
Received 25 January 2002; received in revised form 19 April 2002; accepted 21 May 2002
Abstract The fundamental role of dendritic cells (DC) in initiating and directing the primary immune response is well established. Furthermore, it is now accepted that DC may be useful in new vaccination strategies for preventing certain malignant and infectious diseases. As blood DC (BDC) physiology differs from that of the DC homologues generated in vitro from monocyte precursors, it is becoming more relevant to consider BDC for therapeutic interventions. Until recently, protocols for the isolation of BDC were laborious and inefficient; therefore, their use for investigative cancer immunotherapy is not widespread. In this study, we carefully documented BDC counts, yields and subsets during apheresis (Cobe Spectra), the initial and essential procedure in creating a BDC isolation platform for cancer immunotherapy. We established that an automated software package (Version 6.0 AutoPBPC) provides an operator-independent reliable source of mononuclear cells (MNC) for BDC preparation. Further, we observed that BDC might be recovered in high yields, often greater than 100% relative to the number of circulating BDC predicted by blood volume. An average of 66 million (range, 17 – 179) BDC per 10-l procedure were obtained, largely satisfying the needs for immunization. Higher yields were possible on total processed blood volumes of 15 l. BDC were not activated by the isolation procedure and, more importantly, both BDC subsets (CD11c+CD123low and CD11c CD123high) were equally represented. Finally, we established that the apheresis product could be used for antibody-based BDC immunoselection and demonstrated that fully functional BDC can be obtained by this procedure. D 2002 Published by Elsevier Science B.V. Keywords: Immunotherapy; Dendritic cells; Apheresis; Isolation
Abbreviations: APC, antigen-presenting cell; BDC, Blood DC; DC, dendritic cells; MNC, mononuclear cells; Mo-DC, monocyte-derived DC; PBMC, peripheral blood mononuclear cells; SCF, stem cell factor; TBV, total blood volume. * Corresponding author. Tel.: +61-7-3840-2557; fax: +61-7-3840-2550. E-mail address: [email protected] (D.N.J. Hart). 0022-1759/02/$ - see front matter D 2002 Published by Elsevier Science B.V. PII: S 0 0 2 2 - 1 7 5 9 ( 0 2 ) 0 0 1 8 5 - 0
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1. Introduction Dendritic cells (DC) due to their unique properties as initiators and modulators of the immune system have become the focus of studies for the development of vaccination strategies for malignant disease (Hart and Hill, 1999; Fong and Engleman, 2000). Emerging data from Phase I and Phase II studies show very promising results (Hsu et al., 1996; Nestle et al., 1997; Murphy et al., 1999; Geiger et al., 2000; Kugler et al., 2000). However, there is a lack of consensus with respect to the methods of DC isolation, the source cell population for derivation of DC, the need for and influence of exogenous cytokine stimulation and the effects of the isolation strategy on the DC product (Nestle et al., 2001). It is also now important to employ Good Manufacturing Practice principles to ensure consistency of the DC product and compliance with an increasingly stringent regulatory environment. DC plays a fundamental role both in innate and cognate immune responses (Hart, 1997; Clark et al., 2000). They circulate in blood as precursors with an intermediate differentiation/activation phenotype and represent a small (less than 1%) proportion of blood mononuclear cells (MNC). DC moves from the circulation into the tissues, where they acquire a fully mature antigen-presenting cell (APC) phenotype after interaction with pathogens or other stimuli. This is characterized by the expression of key surface molecules such as CD40, CD80 and CD86, which are involved in costimulation as well as other markers of DC differentiation/activation (Banchereau et al., 2000; Hart et al., 2000). Various subsets of DC have been described to date and they appear to have different physiological functions (Rissoan et al., 1999); they are defined by the expression of the CD123 (IL-3 receptor) and the CD11c antigens (Olweus et al., 1997; Robinson et al., 1999). Various approaches have been used to achieve DC isolation resulting in differences in the properties of the generated DC preparations (Ferlazzo et al., 1999). DClike cells can be produced via transformation of blood monocytes in vitro in the presence of various cytokines and are referred to as monocyte-derived DC (Mo-DC) (Romani et al., 1994; Sallusto and Lanzavecchia, 1994); additionally, DC can be expanded from CD34+ progenitors obtained from bone marrow (Reid et al., 1992; Egner et al., 1993; Szabolcs et al., 1995;
Young et al., 1995), peripheral blood (Mackensen et al., 1995; Siena et al., 1995; Strunk et al., 1996; Monji et al., in press) or neonatal cord blood (Caux et al., 1992, 1996). Alternatively, DC and their precursors present in the blood (blood dendritic cells, BDC) can be enriched using density gradients (Mehta-Damani et al., 1994; McLellan et al., 1995) and selected further using monoclonal antibodies (Fearnley et al., 1997; Schakel et al., 1998; Dzionek et al., 2000). This latter approach encompasses potential theoretical advantages over cytokine-mediated DC in as much as it yields homogenous DC in a defined state of differentiation, capable of responding to physiological stimuli and free from the influence of exogenous cytokines (Hart and Hill, 1999). We have observed differences between BDC and Mo-DC in their expression of surface markers such as DC-SIGN, DEC-205 and the mannose receptor (Hart et al., 2000; Kato et al., 2000). More importantly, BDC appeared to be more efficient in the induction of in vitro primary immune responses (Osugi et al., in press. Blood, 2002), as well as in vitro antigen uptake and processing (Ho et al., 2002). Apheresis products have been used previously as a source of BDC for cancer immunotherapy (Fong et al., 2001), but no detailed studies on BDC phenotype and yields have been published to date. Furthermore, as the phenotype and function (e.g. adhesion molecules and chemokine receptors) of BDC can be easily modulated, it is important to investigate the effect of apheresis on BDC phenotype and function. We have demonstrated previously that BDC numbers can be rapidly increased as a result of external modulators such as surgical and physical stress (Ho et al., 2001) and it is possible that BDC might ‘‘mobilize’’ during the procedure. Given the potential advantages of BDC, we have developed a method for the generation of DC from the mononuclear cell apheresis product via a single magnetic bead separation procedure using the monoclonal antibodies CMRF-44 (Hock et al., 1994; Fearnley et al., 1997) and 56 (Hock et al., 1999). Further description of the kinetics of CMRF-44/56 expression on BDL and their purification will be provided elsewhere (Lopez et al., submitted JIM, 2002). In order to improve BDC yields and maximize their use in cancer vaccination, it is essential to determine optimal apheresis conditions. We describe a clinically applicable technique using apheresis of mononuclear cells from
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unstimulated healthy donors for the purification of BDC from the apheresis product via a single magnetic bead separation procedure.
2. Materials and methods 2.1. Study population The Mater Misericordiae Health Services Ethics Committee approved the study. Healthy volunteers (n=14; 10 males, 4 females), average age 41 (range 21 –56), participated in this study. Medical evaluation and complete blood cell counts were performed prior to apheresis. No prior cytokine stimulation/mobilization was attempted. 2.2. Apheresis isolation software Two different programs on the Cobe Spectra (Gambro BCT, Lakewood CO, USA) were compared for BDC collection. The semi-automated (Version 4.7
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software MNC Program) ‘‘manual’’ version, with variable inlet flow rate from 20 to 150 ml/min allows a continuous collection of the mononuclear cell (MNC) layer forming at the interface between the red cells and the plasma. The product is collected when the operator judges the interface position to be optimal based on collect line appearance (judged by comparison with the Spectra WBC colorgram). The hematocrit range of the collected products was 3 –5%. This protocol takes into account hematocrit, sex, height and weight to determine the flow rates required. The second protocol, the automated (Version 6.1 software, AutoPBPC ) version, accumulates MNC at the interface position and periodically harvests them according to the rate of movement of MNC into the centrifuge channel. Input of subject MNC count, hematocrit, height, weight and sex enables the Spectra software to automatically control the collection over three phases: accumulation, harvest and chase. We configured the system to harvest 4-ml volumes of MNC and a platelet-rich plasma chase of 7- or 9-ml volumes.
Fig. 1. FACS profile of MNC labeled and analyzed for DC. Forward and side scatter profile selected viable cells (left panel) and live cells were labeled with lineage markers and HLA-DR+. Lin HLA-DR+ cells (region R2 in middle panel) were analyzed for BDC subsets in dot plots (right panel).
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2.3. Apheresis sample collection Peripheral venous blood samples (20 ml) were taken from a peripheral vein before apheresis (time 0), 30 min, 24 and 48 h after completion of the procedure. Apheresis was performed in the Mater Hospital Clin-
ical Apheresis Unit. Additionally, at various time points during the procedure, the products corresponding to total processed blood volumes of 2.5, 5, 7.5, 10, 12.5 and 15 l were collected. Unless specifically stated, samples were harvested at every planned volume, except when no product had been collected by the
Fig. 2. Selection of harvesting software protocol. Data corresponding to seven experiments run with the automatic protocol Spectra Version 6.1, AutoPBPC (shown in open bars, 1, 4, 10, 16, 19, 21 and 22) and four experiments run with the manual protocol Spectra Version 4.7, MNC (shown in full bars, 2, 3, 9 and 20). (A) Bars show total MNC, volumes, platelets and DC recovered in 10-l apheresis products for each experiment. (B) BDC efficiency of recovery in repeated experiments performed in three different donors, using the two software options. Placed horizontally, experiments performed on the same individual with a minimum of 2 weeks interval. Calculated BDC (106) present in TBV before the procedure (as described in Materials and methods) were compared with the total BDC recovered after apheresis and expressed as total DC recovered.
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machine. In some experiments, samples were taken from the access vein immediately before each product harvest. ACD-A was used as anticoagulant at a ratio of 1:12 to the inlet flow. Normal healthy volunteers participated in the study and they received standard clinical apheresis care. The Mater Adults Hospital’s Ethical Committee approved their participation. 2.4. Isolation of peripheral blood mononuclear cells (PBMC) and labeling of BDC MNC were isolated by Ficoll– Hypaque (Pharmacia, Sweden) density gradient and the interphase cells washed three times. Cells were labeled in a 50-Al mixture of hybridoma supernatants containing antibodies specific for: CD14 (CMRF-31, IgG2a; produced in this laboratory), CD3 (OKT3, IgG2a, American Type Tissue Collection, Rockville, MD), CD16 (HuNK2, IgG2a) and CD19 (FMC63, IgG1), gifts of Prof H. Zola, Adelaide, Australia. After one washing step, fluorescein-conjugated sheep – mouse immunoglobulin-specific (FITC-SAM, Amrad Biotech, Victoria, Australia) was added, followed by saturating concentrations of mouse immunoglobulin (Sigma, St. Louis). Finally, PE.Cyanin5-conjugated HLA-DR specific antibody (Immu375, IgG1, Immunotech, Marseille, France) was added together with either PE-conjugated CD11c (S-HCL-3, IgG2a, BD Bioscience, BDIS, San Jose, CA) or PE-conjugated CD123 (a chain specific, 9F5, IgG2b, PharMingen International, San Diego, CA). IgG1 (MOPC-21), IgG2a (G155-178) and IgG2b (27-35) isotype controls were purchased from PharMingen. For certain experiments, the following antibodies were used: PEconjugated CD14 (leuM3, IgG2b), CD19 (leuM12, IgG1) and CD80 (L307.4, IgG1) from BDIS and PEconjugated CD40 (5C3, IgG1), CD86 (24F, IgG1, PharMingen). Labeled samples were analyzed by FACSCalibur flow cytometer (BDIS). 2.5. DC counts BDC counts (expressed as 106/l) were calculated from the number of MNC/L blood (determined by the automated cell counter Advia 120, Hematology System, Bayer, Tarrytown, NY) multiplied by the percentage of BDC (Ho et al., 2001). Alternatively, values were also expressed as the total content of BDC
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calculated on total blood volume (TBV) of every volunteer. 2.6. DC isolation The BDC purification procedure will be described elsewhere (Lopez et al., submitted. JIM, 2002). Briefly, magnetic bead separation of BDC was undertaken using biotinylated CMRF-44 (Hock et al., 1994) antibody and biotin-specific beads (Miltenyi Biotech, Bergisch Gladbach, Germany). Positive and negative fractions were collected and evaluated by flow cytometry (level of BDC enrichment) and MLR (function). For the experiment with frozen cells, apheresis material was diluted in ice-cold DMEM medium to 50% of the total final volume and supplemented with 10% DMSO and 40% AB pooled serum, stored at 70 jC in an insulated styrofoam container and tested after 2 weeks of storage. 2.7. MLR with isolated BDC A titration of irradiated MNC (cultured and fresh), CMRF-44+ magnetically selected BDC or the negative fraction of the CMRF-44 isolation (5103 – 3105 cells) was incubated with allogeneic T-lymTable 1 Efficiency of recovery of BDC: blood dendritic cells (BDC) recovered after apheresis expressed as a percentage of total blood volume (TBV)a Experiment
Software used Automatic
1 2 3 4 9 10 16 19 20 21 22
Manual
179 126 50 120 44 23 28 65 65 40 101
Mean SE a
79.4 21.6
71.3 18.8
Percentage was established on the basis of the TBV (before starting the procedure) and the calculated total DC recovered in the apheresis product.
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phocytes (5104 cells) for 5 days in 96-well Ubottom plates. Sixteen hours prior to harvesting the cells, 0.5 ACi of 3H-thymidine was added to each well. 3H-thymidine uptake was counted in a liquid hscintillation counter (Wallac, MicroBeta Trilux Scintillation Counter, Turku, Finland). 2.8. Statistical analysis Paired statistical analysis was performed using the Student’s two-tailed t-test.
3. Results 3.1. Definition of BDC for counting Quantification of BDC was performed defining class II expressing cells, lacking markers for specific hemopoietic lineages. DC subsets were identified as CD11c+ or CD123+ cells within the BDC gated population (Fig. 1). For the purpose of calculations, BDC were defined as the percentage of lineage negative HLA-DR high expressing cells.
Fig. 3. Total BDC recovery. (A) Detailed evaluation of BDC present in 10-l (open bars) and 15-l apheresis (hatched) products; the numbers of BDC (106) present at various harvesting points are shown as independent bars (right axis) and the percentages of BDC as a line (left axis). (B) Percentage of BDC present in blood before, 0.5, 24 and 48 h after the procedure.
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Fig. 3 (continued).
3.2. Compliance with the apheresis procedure As reported in the literature (Furuta et al., 1999; Sato et al., 2001), apheresis was safe and well tolerated. Of the 24 procedures undertaken, 1 was aborted due to difficult venous access and 1 due to mild hypocalcaemia symptoms (spontaneously reverting); the rest were well tolerated with only local discomfort that ceased at the end of the procedure. Only a small (average 13%) reduction in the platelet counts was detected at the end of the apheresis (mean 235109/ml before and mean 201109/ml after). 3.3. Optimal software for BDC isolation Ten-liter collections were performed using the semi-automated (Version 4.7 software MNC Program) ‘‘manual’’ or fully ‘‘automatic’’ (AutoPBSC) software versions to control the COBE Spectra. A comparison of the volume, total of MNC recovered, number of
platelets and total BDC recovered in the apheresis product is shown in Fig. 2A. The automatic software (n=7) resulted in consistently lower product volumes (meanFS.E.=automatic: 45F5 ml versus manual: 116F8 ml; p<0.005) and the number of contaminating platelets was also significantly lower than in the manual (n=4) collections (automatic: 57F10109 versus manual: 459F104109; p<0.05). Although the total number of MNC recovered from the manual procedures was, as expected, higher than that obtained with the automatic software (11.088F1.415109 and 5.982F1.148109, respectively; p<0.05), there was no statistically significant difference in the number of BDC recovered (automatic: 69F16106 versus manual: 102F35106; p=0.34). Furthermore, the efficiency to recover the available circulating BDC (predicted blood volumeBDC count) was comparable using the two software protocols (automatic: 80F22% versus manual: 71F19%; p=0.77). As shown in Table 1, the efficiency of recovery differed
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Fig. 4. Efficiency of BDC recovery. (A) The total BDC (106) present in the apheresis product from 10-l (open bars) and 15-l (shaded bars) apheresis. (B) The percentage of the theoretical total BDC available obtained based on TBV is plotted.
considerably between individual experiments possibly as a reflection of the variations in BDC counts documented previously (Ho et al., 2001). Three sets of experiments were performed on the same individuals utilizing automatic and manual software with a minimum 2-week interval (experiments 1 and 2; 10,19 and 9; 21,22 and 20). In this group of experiments (Fig. 2B), the automatic software (experiments 1, 10, 19, 21 and 22) produced an average 82% BDC recovery as opposed to 78% with the manual procedure (experiments 2, 9 and 20). The automatic software was considered advantageous because of the smaller product volume, lower platelet contamination
and reduced operator dependency, appropriate for multicenter trials. 3.4. Optimal harvesting volume We next evaluated the recovery of BDC obtained using the automatic software at different total inlet volumes. Firstly, we studied four 10-l and five 15-l
Table 2 Activation status of blood dendritic (BDC) before, during and after apheresis procedure: percentage of BDC Sample
% CD40+
% CD80+
%CD86+
Experiment 2
3
4
2
3
4
2
3
4
Before 7 47.4 25.4 0 4.5 1.1 0.97 38.8 57.5 Product 14 23.5 37.6 0.12 5.0 2.9 1.33 41.5 39.0 After 30 min 6 57.3 22.8 0 3.6 1.2 0.33 34.2 56.7 24 h 11.9 0.5 44.5 48 h 21.2 8.4 0.5 0.3 43.9 62.5
Fig. 5. BDC subset distribution in apheresis product. MeanFS.E. of the percentage of CD11c+ and CD123+ in blood samples before and after apheresis procedure (n=6, experiments 1, 2, 3, 4, 9 and 10) is shown.
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procedures in detail. As shown in Fig. 3A, more than 50% of all the BDC recovery took place during the later part of the procedure; this observation was made both in the 10-l and the 15-l procedures. The higher yields towards the end of the procedure resulted from the combined effect of a slight increase in the percentage of BDC (Fig. 3A, lines) and an increase in the MNC in the product (not shown). There were only minor changes in the percentage of BDC in blood after the procedure. Although there was not a defined pattern in all of the experiments, small decreases in BDC% were detected 30 min after the
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end of the procedure with a fast rebound at 24 h in experiments 6, 8, 13 and 17 (Fig. 3B), i.e. four of nine procedures. 3.5. Efficiency of BDC recovery The efficiency of BDC recovery by the automatic procedure was evaluated further by extending the number of volunteers. As shown in Fig. 4A, the efficiency of recovery was variable (n=17; mean efficiency 73%; range 17 – 179%) and it reflected volunteer variations. Recoveries that were greater than
Fig. 6. Isolation of CMRF-44 BDC from apheresis product. Dot plots show FACS analysis of cells before magnetic bead separation (upper panels), in negative (middle panels) and positive (lower panels) fractions. Percentage of cells present in every quadrant of the dot plot is shown. Left panels: experiments with freshly obtained apheresis product. Right panels: results obtained with the same cells frozen and thawed after 2 weeks.
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100% of the predicted number of BDC in TBV indicated possible BDC mobilization during the procedures. Furthermore, the finding reinforced this observation that higher recoveries were observed at the latter harvesting points (data not shown). The yield from the 5 15-l apheresis procedure was 112F21106 (meanFS.E.), while that of the 12 10-l procedure was 78F17106 ( p=0.2999). The efficiency of the 15-l procedures was 97F10% and that of the 10-l procedure, 66F14% ( p=0.202). Evaluation of the BDC percentage and BDC counts in peripheral blood obtained from the access line was performed at times coinciding with the different apheresis volume harvests. These remained constant throughout the procedure (data not shown). This finding was also observed in the 15-l experiments. These data further reinforce the point that active BDC mobilization or recruitment occurred during the apheresis. 3.6. BDC costimulatory phenotype is not altered by the apheresis procedure As shown in Table 2, the apheresis procedure does not generate significant changes in the expression of CD40, CD80 and CD86 on lineage HLA-DR+ DC (n=3). 3.7. DC subset distribution remains unchanged after apheresis The BDC subset proportions remained unchanged after the procedure. Fig. 5, summarizes the percentage CD11c+ DC and CD123+ subset distribution observed in six experiments. In these experiments, the CD11c+/CD123+ DC ratio before apheresis was 0.52F0.17 versus 0.45F0.18 in the product ( p= 0.502). 3.8. BDC can be successfully isolated from apheresis product We have optimized an isolation protocol for BDC, based on the use of the CMRF-44 monoclonal antibody and magnetic bead technology to purify BDC after overnight culture (Lopez et al., submitted for publication). Following apheresis, CMRF-44 antigen expression in vitro was not altered and purification
Fig. 7. Allogeneic MLR evaluation of enriched BDC prepared from apheresis MNC. Freshly harvested apheresis cells (fresh) were cultured overnight (cultured), labeled with CMRF-44 biotinylated antibody and enriched with a positive immunoselection column. Both positive and negative fraction cells were collected and tested. A titration of APC from each origin was cultured with isolated allogeneic T-cells, as described in Materials and methods.
of the BDC was readily achieved (Fig. 6). Furthermore, the separation of BDC from both fresh and frozen apheresis products yielded comparable BDC purity (Fig. 6). Furthermore, repeated BDC isolations (n=4) performed on the same frozen apheresis material yielded very consistent results (data not shown). The BDC purified in this way from apheresis material were fully functional in an allogeneic MLR (Fig. 7).
4. Discussion Clinical use of DC requires a highly reproducible and GMP applicable method that provides sufficient yields of nonactivated cells that are fully functional upon cryopreservation. Here, we evaluated the use of two software protocols for the collection of BDC using the Cobe Spectra apheresis machine and established that the automated version (Auto 6.1) provides a lesser volume product and reduced platelet contaminant. We also demonstrated that the apheresis product
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contains sufficient numbers of BDC in a nonactivated state suitable for immunotherapy. No skewing of subset composition was observed allowing further subset fractionation, if appropriate. The data suggests that BDC are mobilized during the procedure and although a 10-l apheresis procedure is sufficient, if necessary, a 15-l apheresis procedure will generate an improved BDC yield. Finally, we showed that BDC can be isolated from the apheresis product and that they are also fully functional after cryopreservation. A detailed functional evaluation of BDC purified after a magnetic bead separation following apheresis is presented elsewhere (Lopez et al., in preparation). The selection of an automated protocol for the harvest of BDC has various advantages. The reproducibility and the operator independence permits the standardization of the method across laboratories, a particularly relevant issue when multi-center protocols are envisaged, as will be important in evaluating DC cancer immunotherapy protocols. Apheresis using automated Cobe AutoPBSC software has also been used to obtain MNC for the preparation of MoDC and these were likewise fully functional; however, details on the apheresis procedure are not available for comparison (Lewalle et al., 2000). Apheresis MNC products have been used successfully for the production of Mo-DC from monocytes (Thurner et al., 1999; Goxe et al., 2000; Lewalle et al., 2000) and Langerhans cells from CD34+ progenitors but again, data on yields were not provided (Gatti et al., 2000). Apheresed MNC preparations have also been used for density gradient separation of BDC but no information has been published regarding the yields (Fong et al., 2001). The reduced product volume facilitates further BDC isolation procedures including the labeling with monoclonal antibodies. The Cobe AutoPBPC program provided lesser platelet contamination of the sample. This feature is particularly relevant, since platelets are known for their capacity to activate BDC; for example, high platelet contamination has been shown to hinder Mo-DC generation from apheresis material both in melanoma patients and healthy volunteers (Glaser et al., 1999). Critical to the clinical use of the pheresed material is the ability to handle the products in a closed clinical grade environment. Clinical grade Mo-DC has been prepared from pheresed MNC after processing with
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the Cobe Spectra cell separator (Rouard et al., 2000). This methodology can now be applied to BDC. The product can then be enriched significantly for BDC, whilst maintaining sterile Good Manufacturing Practice (GMP) procedures, using a BDC-specific mAb for positive cell selection. Cryopreservation of DC preparations will also be highly advantageous for clinical scheduling. Protocols examining the generation of Mo-DC have shown that they may be stored safely frozen either as PBMC or as matured Mo-DC (Thurner et al., 1999; Lewalle et al., 2000). It has also been demonstrated that antigenloaded Mo-DC obtained from apheresis protocols can be stored frozen, retaining their stimulatory function (Feuerstein et al., 2000; Lewalle et al., 2000). Our data shows that BDC can also be obtained from frozen PBMC collected by apheresis. Thawing cells prior to an overnight incubation before the antibody-mediated separation of DC makes this a very convenient protocol. Mo-DC has been generated from the apheresis products obtained from patients with multiple myeloma (Tarte et al., 1997) and chronic myeloid leukemia (Zheng et al., 2000). Indeed, we now have data that establishes the optimal BDC harvesting time in G-CSF and cyclophosphamide-treated patients with non-Hodgkin lymphoma and multiple myeloma, who are undergoing mobilization for blood stem cell collection (Vuckovic et al., in preparation). The data also predict that sufficient numbers of BDC will be obtained from apheresis in those patients without the need of additional mobilization. However, various mobilizing conditions have been used to optimize the generation of monocyte and CD34+-derived DC, resulting in changes in the phenotype and/or function of various cell types including T-lymphocytes NK and DC (Gazitt, 2000; Roth et al., 2000). Stem cell factor (SCF) mobilization in patients with breast cancer resulted in minimal increases in BDC populations (Menedez et al., 2001). Flt-3 ligand mobilizes BDL in healthy volunteers (Maraskovsky et al., 2000) and is currently being investigated by several groups for its ability to mobilize BDC in patients. DC are subject to various physiological influences that may change their status of activation and/or mobilization. We have observed that stress (surgery and extreme exercise) prompts mobilization of BDC without activation (Ho et al., 2001). Likewise, we
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have documented changes in certain disease states (Summers et al., 1999; Vuckovic et al., 1999; Brown et al., 2001). Given their responses to environmental influences, it was not inconceivable that apheresis would change BDC differentiation/activation. Our data on the evaluation of whole DC suggest that the apheresis procedure does not induce the activation state of collected BDC; a highly desirable feature allowing for in vitro optimization of BDC function for immunotherapy protocols. Differential activation of a proportion of cells within certain DC subsets (MacDonald et al., in press) may escape detection and will be the subject of future analyses. In contrast, maturation of Mo-DC from an apheresis product was reported to occur earlier than for cells drawn from fresh blood samples (Thurner et al., 1999). We have shown here that BDC isolated from apheresis MNC preparations exhibited similar functions to BDC drawn by direct venipuncture. In conclusion, this study describes critical information required to generate sufficient, nonactivated and fully functional BDC in a standard manner for DC-based cancer immunotherapy studies.
Acknowledgements We gratefully acknowledge the enthusiastic participation of the volunteers recruited for this study; we thank Drs. Cameron Turtle, Chris Ho, Geoff Hill and Nick Murray for constructive discussions and Gilles Bioley for help with the functional evaluation of apheresis products. We acknowledge the support Miltenyi Biotech (Bergisch Gladbach, Germany) for immuno-isolation reagents. This work was funded by the Mater Medical Research Institute and by GAMBRO BCT (Lakewood CO).
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Monitoring and isolation of blood dendritic cells from apheresis products in healthy individuals: a platform for cancer immunotherapy J. Alejandro Lo´pez a, Georgina Crosbie a, Cathryn Kelly b, Ann Marie McGee a, Katrina Williams b, Slavica Vuckovic a, Robert Schuyler c, Robyn Rodwell b, Sue J. Wright b, Kerry Taylor b, Derek N.J. Hart a,* a
Mater Medical Research Institute, Aubigny Place, South Brisbane 4101, Australia b Mater Adult Hospital, Raymond Terrace, South Brisbane 4101, Australia c Gambro BCT, Lakewood, CO 80215, USA
Received 25 January 2002; received in revised form 19 April 2002; accepted 21 May 2002
Abstract The fundamental role of dendritic cells (DC) in initiating and directing the primary immune response is well established. Furthermore, it is now accepted that DC may be useful in new vaccination strategies for preventing certain malignant and infectious diseases. As blood DC (BDC) physiology differs from that of the DC homologues generated in vitro from monocyte precursors, it is becoming more relevant to consider BDC for therapeutic interventions. Until recently, protocols for the isolation of BDC were laborious and inefficient; therefore, their use for investigative cancer immunotherapy is not widespread. In this study, we carefully documented BDC counts, yields and subsets during apheresis (Cobe Spectra), the initial and essential procedure in creating a BDC isolation platform for cancer immunotherapy. We established that an automated software package (Version 6.0 AutoPBPC) provides an operator-independent reliable source of mononuclear cells (MNC) for BDC preparation. Further, we observed that BDC might be recovered in high yields, often greater than 100% relative to the number of circulating BDC predicted by blood volume. An average of 66 million (range, 17 – 179) BDC per 10-l procedure were obtained, largely satisfying the needs for immunization. Higher yields were possible on total processed blood volumes of 15 l. BDC were not activated by the isolation procedure and, more importantly, both BDC subsets (CD11c+CD123low and CD11c CD123high) were equally represented. Finally, we established that the apheresis product could be used for antibody-based BDC immunoselection and demonstrated that fully functional BDC can be obtained by this procedure. D 2002 Published by Elsevier Science B.V. Keywords: Immunotherapy; Dendritic cells; Apheresis; Isolation
Abbreviations: APC, antigen-presenting cell; BDC, Blood DC; DC, dendritic cells; MNC, mononuclear cells; Mo-DC, monocyte-derived DC; PBMC, peripheral blood mononuclear cells; SCF, stem cell factor; TBV, total blood volume. * Corresponding author. Tel.: +61-7-3840-2557; fax: +61-7-3840-2550. E-mail address: [email protected] (D.N.J. Hart). 0022-1759/02/$ - see front matter D 2002 Published by Elsevier Science B.V. PII: S 0 0 2 2 - 1 7 5 9 ( 0 2 ) 0 0 1 8 5 - 0
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1. Introduction Dendritic cells (DC) due to their unique properties as initiators and modulators of the immune system have become the focus of studies for the development of vaccination strategies for malignant disease (Hart and Hill, 1999; Fong and Engleman, 2000). Emerging data from Phase I and Phase II studies show very promising results (Hsu et al., 1996; Nestle et al., 1997; Murphy et al., 1999; Geiger et al., 2000; Kugler et al., 2000). However, there is a lack of consensus with respect to the methods of DC isolation, the source cell population for derivation of DC, the need for and influence of exogenous cytokine stimulation and the effects of the isolation strategy on the DC product (Nestle et al., 2001). It is also now important to employ Good Manufacturing Practice principles to ensure consistency of the DC product and compliance with an increasingly stringent regulatory environment. DC plays a fundamental role both in innate and cognate immune responses (Hart, 1997; Clark et al., 2000). They circulate in blood as precursors with an intermediate differentiation/activation phenotype and represent a small (less than 1%) proportion of blood mononuclear cells (MNC). DC moves from the circulation into the tissues, where they acquire a fully mature antigen-presenting cell (APC) phenotype after interaction with pathogens or other stimuli. This is characterized by the expression of key surface molecules such as CD40, CD80 and CD86, which are involved in costimulation as well as other markers of DC differentiation/activation (Banchereau et al., 2000; Hart et al., 2000). Various subsets of DC have been described to date and they appear to have different physiological functions (Rissoan et al., 1999); they are defined by the expression of the CD123 (IL-3 receptor) and the CD11c antigens (Olweus et al., 1997; Robinson et al., 1999). Various approaches have been used to achieve DC isolation resulting in differences in the properties of the generated DC preparations (Ferlazzo et al., 1999). DClike cells can be produced via transformation of blood monocytes in vitro in the presence of various cytokines and are referred to as monocyte-derived DC (Mo-DC) (Romani et al., 1994; Sallusto and Lanzavecchia, 1994); additionally, DC can be expanded from CD34+ progenitors obtained from bone marrow (Reid et al., 1992; Egner et al., 1993; Szabolcs et al., 1995;
Young et al., 1995), peripheral blood (Mackensen et al., 1995; Siena et al., 1995; Strunk et al., 1996; Monji et al., in press) or neonatal cord blood (Caux et al., 1992, 1996). Alternatively, DC and their precursors present in the blood (blood dendritic cells, BDC) can be enriched using density gradients (Mehta-Damani et al., 1994; McLellan et al., 1995) and selected further using monoclonal antibodies (Fearnley et al., 1997; Schakel et al., 1998; Dzionek et al., 2000). This latter approach encompasses potential theoretical advantages over cytokine-mediated DC in as much as it yields homogenous DC in a defined state of differentiation, capable of responding to physiological stimuli and free from the influence of exogenous cytokines (Hart and Hill, 1999). We have observed differences between BDC and Mo-DC in their expression of surface markers such as DC-SIGN, DEC-205 and the mannose receptor (Hart et al., 2000; Kato et al., 2000). More importantly, BDC appeared to be more efficient in the induction of in vitro primary immune responses (Osugi et al., in press. Blood, 2002), as well as in vitro antigen uptake and processing (Ho et al., 2002). Apheresis products have been used previously as a source of BDC for cancer immunotherapy (Fong et al., 2001), but no detailed studies on BDC phenotype and yields have been published to date. Furthermore, as the phenotype and function (e.g. adhesion molecules and chemokine receptors) of BDC can be easily modulated, it is important to investigate the effect of apheresis on BDC phenotype and function. We have demonstrated previously that BDC numbers can be rapidly increased as a result of external modulators such as surgical and physical stress (Ho et al., 2001) and it is possible that BDC might ‘‘mobilize’’ during the procedure. Given the potential advantages of BDC, we have developed a method for the generation of DC from the mononuclear cell apheresis product via a single magnetic bead separation procedure using the monoclonal antibodies CMRF-44 (Hock et al., 1994; Fearnley et al., 1997) and 56 (Hock et al., 1999). Further description of the kinetics of CMRF-44/56 expression on BDL and their purification will be provided elsewhere (Lopez et al., submitted JIM, 2002). In order to improve BDC yields and maximize their use in cancer vaccination, it is essential to determine optimal apheresis conditions. We describe a clinically applicable technique using apheresis of mononuclear cells from
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unstimulated healthy donors for the purification of BDC from the apheresis product via a single magnetic bead separation procedure.
2. Materials and methods 2.1. Study population The Mater Misericordiae Health Services Ethics Committee approved the study. Healthy volunteers (n=14; 10 males, 4 females), average age 41 (range 21 –56), participated in this study. Medical evaluation and complete blood cell counts were performed prior to apheresis. No prior cytokine stimulation/mobilization was attempted. 2.2. Apheresis isolation software Two different programs on the Cobe Spectra (Gambro BCT, Lakewood CO, USA) were compared for BDC collection. The semi-automated (Version 4.7
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software MNC Program) ‘‘manual’’ version, with variable inlet flow rate from 20 to 150 ml/min allows a continuous collection of the mononuclear cell (MNC) layer forming at the interface between the red cells and the plasma. The product is collected when the operator judges the interface position to be optimal based on collect line appearance (judged by comparison with the Spectra WBC colorgram). The hematocrit range of the collected products was 3 –5%. This protocol takes into account hematocrit, sex, height and weight to determine the flow rates required. The second protocol, the automated (Version 6.1 software, AutoPBPC ) version, accumulates MNC at the interface position and periodically harvests them according to the rate of movement of MNC into the centrifuge channel. Input of subject MNC count, hematocrit, height, weight and sex enables the Spectra software to automatically control the collection over three phases: accumulation, harvest and chase. We configured the system to harvest 4-ml volumes of MNC and a platelet-rich plasma chase of 7- or 9-ml volumes.
Fig. 1. FACS profile of MNC labeled and analyzed for DC. Forward and side scatter profile selected viable cells (left panel) and live cells were labeled with lineage markers and HLA-DR+. Lin HLA-DR+ cells (region R2 in middle panel) were analyzed for BDC subsets in dot plots (right panel).
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2.3. Apheresis sample collection Peripheral venous blood samples (20 ml) were taken from a peripheral vein before apheresis (time 0), 30 min, 24 and 48 h after completion of the procedure. Apheresis was performed in the Mater Hospital Clin-
ical Apheresis Unit. Additionally, at various time points during the procedure, the products corresponding to total processed blood volumes of 2.5, 5, 7.5, 10, 12.5 and 15 l were collected. Unless specifically stated, samples were harvested at every planned volume, except when no product had been collected by the
Fig. 2. Selection of harvesting software protocol. Data corresponding to seven experiments run with the automatic protocol Spectra Version 6.1, AutoPBPC (shown in open bars, 1, 4, 10, 16, 19, 21 and 22) and four experiments run with the manual protocol Spectra Version 4.7, MNC (shown in full bars, 2, 3, 9 and 20). (A) Bars show total MNC, volumes, platelets and DC recovered in 10-l apheresis products for each experiment. (B) BDC efficiency of recovery in repeated experiments performed in three different donors, using the two software options. Placed horizontally, experiments performed on the same individual with a minimum of 2 weeks interval. Calculated BDC (106) present in TBV before the procedure (as described in Materials and methods) were compared with the total BDC recovered after apheresis and expressed as total DC recovered.
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machine. In some experiments, samples were taken from the access vein immediately before each product harvest. ACD-A was used as anticoagulant at a ratio of 1:12 to the inlet flow. Normal healthy volunteers participated in the study and they received standard clinical apheresis care. The Mater Adults Hospital’s Ethical Committee approved their participation. 2.4. Isolation of peripheral blood mononuclear cells (PBMC) and labeling of BDC MNC were isolated by Ficoll– Hypaque (Pharmacia, Sweden) density gradient and the interphase cells washed three times. Cells were labeled in a 50-Al mixture of hybridoma supernatants containing antibodies specific for: CD14 (CMRF-31, IgG2a; produced in this laboratory), CD3 (OKT3, IgG2a, American Type Tissue Collection, Rockville, MD), CD16 (HuNK2, IgG2a) and CD19 (FMC63, IgG1), gifts of Prof H. Zola, Adelaide, Australia. After one washing step, fluorescein-conjugated sheep – mouse immunoglobulin-specific (FITC-SAM, Amrad Biotech, Victoria, Australia) was added, followed by saturating concentrations of mouse immunoglobulin (Sigma, St. Louis). Finally, PE.Cyanin5-conjugated HLA-DR specific antibody (Immu375, IgG1, Immunotech, Marseille, France) was added together with either PE-conjugated CD11c (S-HCL-3, IgG2a, BD Bioscience, BDIS, San Jose, CA) or PE-conjugated CD123 (a chain specific, 9F5, IgG2b, PharMingen International, San Diego, CA). IgG1 (MOPC-21), IgG2a (G155-178) and IgG2b (27-35) isotype controls were purchased from PharMingen. For certain experiments, the following antibodies were used: PEconjugated CD14 (leuM3, IgG2b), CD19 (leuM12, IgG1) and CD80 (L307.4, IgG1) from BDIS and PEconjugated CD40 (5C3, IgG1), CD86 (24F, IgG1, PharMingen). Labeled samples were analyzed by FACSCalibur flow cytometer (BDIS). 2.5. DC counts BDC counts (expressed as 106/l) were calculated from the number of MNC/L blood (determined by the automated cell counter Advia 120, Hematology System, Bayer, Tarrytown, NY) multiplied by the percentage of BDC (Ho et al., 2001). Alternatively, values were also expressed as the total content of BDC
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calculated on total blood volume (TBV) of every volunteer. 2.6. DC isolation The BDC purification procedure will be described elsewhere (Lopez et al., submitted. JIM, 2002). Briefly, magnetic bead separation of BDC was undertaken using biotinylated CMRF-44 (Hock et al., 1994) antibody and biotin-specific beads (Miltenyi Biotech, Bergisch Gladbach, Germany). Positive and negative fractions were collected and evaluated by flow cytometry (level of BDC enrichment) and MLR (function). For the experiment with frozen cells, apheresis material was diluted in ice-cold DMEM medium to 50% of the total final volume and supplemented with 10% DMSO and 40% AB pooled serum, stored at 70 jC in an insulated styrofoam container and tested after 2 weeks of storage. 2.7. MLR with isolated BDC A titration of irradiated MNC (cultured and fresh), CMRF-44+ magnetically selected BDC or the negative fraction of the CMRF-44 isolation (5103 – 3105 cells) was incubated with allogeneic T-lymTable 1 Efficiency of recovery of BDC: blood dendritic cells (BDC) recovered after apheresis expressed as a percentage of total blood volume (TBV)a Experiment
Software used Automatic
1 2 3 4 9 10 16 19 20 21 22
Manual
179 126 50 120 44 23 28 65 65 40 101
Mean SE a
79.4 21.6
71.3 18.8
Percentage was established on the basis of the TBV (before starting the procedure) and the calculated total DC recovered in the apheresis product.
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phocytes (5104 cells) for 5 days in 96-well Ubottom plates. Sixteen hours prior to harvesting the cells, 0.5 ACi of 3H-thymidine was added to each well. 3H-thymidine uptake was counted in a liquid hscintillation counter (Wallac, MicroBeta Trilux Scintillation Counter, Turku, Finland). 2.8. Statistical analysis Paired statistical analysis was performed using the Student’s two-tailed t-test.
3. Results 3.1. Definition of BDC for counting Quantification of BDC was performed defining class II expressing cells, lacking markers for specific hemopoietic lineages. DC subsets were identified as CD11c+ or CD123+ cells within the BDC gated population (Fig. 1). For the purpose of calculations, BDC were defined as the percentage of lineage negative HLA-DR high expressing cells.
Fig. 3. Total BDC recovery. (A) Detailed evaluation of BDC present in 10-l (open bars) and 15-l apheresis (hatched) products; the numbers of BDC (106) present at various harvesting points are shown as independent bars (right axis) and the percentages of BDC as a line (left axis). (B) Percentage of BDC present in blood before, 0.5, 24 and 48 h after the procedure.
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Fig. 3 (continued).
3.2. Compliance with the apheresis procedure As reported in the literature (Furuta et al., 1999; Sato et al., 2001), apheresis was safe and well tolerated. Of the 24 procedures undertaken, 1 was aborted due to difficult venous access and 1 due to mild hypocalcaemia symptoms (spontaneously reverting); the rest were well tolerated with only local discomfort that ceased at the end of the procedure. Only a small (average 13%) reduction in the platelet counts was detected at the end of the apheresis (mean 235109/ml before and mean 201109/ml after). 3.3. Optimal software for BDC isolation Ten-liter collections were performed using the semi-automated (Version 4.7 software MNC Program) ‘‘manual’’ or fully ‘‘automatic’’ (AutoPBSC) software versions to control the COBE Spectra. A comparison of the volume, total of MNC recovered, number of
platelets and total BDC recovered in the apheresis product is shown in Fig. 2A. The automatic software (n=7) resulted in consistently lower product volumes (meanFS.E.=automatic: 45F5 ml versus manual: 116F8 ml; p<0.005) and the number of contaminating platelets was also significantly lower than in the manual (n=4) collections (automatic: 57F10109 versus manual: 459F104109; p<0.05). Although the total number of MNC recovered from the manual procedures was, as expected, higher than that obtained with the automatic software (11.088F1.415109 and 5.982F1.148109, respectively; p<0.05), there was no statistically significant difference in the number of BDC recovered (automatic: 69F16106 versus manual: 102F35106; p=0.34). Furthermore, the efficiency to recover the available circulating BDC (predicted blood volumeBDC count) was comparable using the two software protocols (automatic: 80F22% versus manual: 71F19%; p=0.77). As shown in Table 1, the efficiency of recovery differed
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Fig. 4. Efficiency of BDC recovery. (A) The total BDC (106) present in the apheresis product from 10-l (open bars) and 15-l (shaded bars) apheresis. (B) The percentage of the theoretical total BDC available obtained based on TBV is plotted.
considerably between individual experiments possibly as a reflection of the variations in BDC counts documented previously (Ho et al., 2001). Three sets of experiments were performed on the same individuals utilizing automatic and manual software with a minimum 2-week interval (experiments 1 and 2; 10,19 and 9; 21,22 and 20). In this group of experiments (Fig. 2B), the automatic software (experiments 1, 10, 19, 21 and 22) produced an average 82% BDC recovery as opposed to 78% with the manual procedure (experiments 2, 9 and 20). The automatic software was considered advantageous because of the smaller product volume, lower platelet contamination
and reduced operator dependency, appropriate for multicenter trials. 3.4. Optimal harvesting volume We next evaluated the recovery of BDC obtained using the automatic software at different total inlet volumes. Firstly, we studied four 10-l and five 15-l
Table 2 Activation status of blood dendritic (BDC) before, during and after apheresis procedure: percentage of BDC Sample
% CD40+
% CD80+
%CD86+
Experiment 2
3
4
2
3
4
2
3
4
Before 7 47.4 25.4 0 4.5 1.1 0.97 38.8 57.5 Product 14 23.5 37.6 0.12 5.0 2.9 1.33 41.5 39.0 After 30 min 6 57.3 22.8 0 3.6 1.2 0.33 34.2 56.7 24 h 11.9 0.5 44.5 48 h 21.2 8.4 0.5 0.3 43.9 62.5
Fig. 5. BDC subset distribution in apheresis product. MeanFS.E. of the percentage of CD11c+ and CD123+ in blood samples before and after apheresis procedure (n=6, experiments 1, 2, 3, 4, 9 and 10) is shown.
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procedures in detail. As shown in Fig. 3A, more than 50% of all the BDC recovery took place during the later part of the procedure; this observation was made both in the 10-l and the 15-l procedures. The higher yields towards the end of the procedure resulted from the combined effect of a slight increase in the percentage of BDC (Fig. 3A, lines) and an increase in the MNC in the product (not shown). There were only minor changes in the percentage of BDC in blood after the procedure. Although there was not a defined pattern in all of the experiments, small decreases in BDC% were detected 30 min after the
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end of the procedure with a fast rebound at 24 h in experiments 6, 8, 13 and 17 (Fig. 3B), i.e. four of nine procedures. 3.5. Efficiency of BDC recovery The efficiency of BDC recovery by the automatic procedure was evaluated further by extending the number of volunteers. As shown in Fig. 4A, the efficiency of recovery was variable (n=17; mean efficiency 73%; range 17 – 179%) and it reflected volunteer variations. Recoveries that were greater than
Fig. 6. Isolation of CMRF-44 BDC from apheresis product. Dot plots show FACS analysis of cells before magnetic bead separation (upper panels), in negative (middle panels) and positive (lower panels) fractions. Percentage of cells present in every quadrant of the dot plot is shown. Left panels: experiments with freshly obtained apheresis product. Right panels: results obtained with the same cells frozen and thawed after 2 weeks.
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100% of the predicted number of BDC in TBV indicated possible BDC mobilization during the procedures. Furthermore, the finding reinforced this observation that higher recoveries were observed at the latter harvesting points (data not shown). The yield from the 5 15-l apheresis procedure was 112F21106 (meanFS.E.), while that of the 12 10-l procedure was 78F17106 ( p=0.2999). The efficiency of the 15-l procedures was 97F10% and that of the 10-l procedure, 66F14% ( p=0.202). Evaluation of the BDC percentage and BDC counts in peripheral blood obtained from the access line was performed at times coinciding with the different apheresis volume harvests. These remained constant throughout the procedure (data not shown). This finding was also observed in the 15-l experiments. These data further reinforce the point that active BDC mobilization or recruitment occurred during the apheresis. 3.6. BDC costimulatory phenotype is not altered by the apheresis procedure As shown in Table 2, the apheresis procedure does not generate significant changes in the expression of CD40, CD80 and CD86 on lineage HLA-DR+ DC (n=3). 3.7. DC subset distribution remains unchanged after apheresis The BDC subset proportions remained unchanged after the procedure. Fig. 5, summarizes the percentage CD11c+ DC and CD123+ subset distribution observed in six experiments. In these experiments, the CD11c+/CD123+ DC ratio before apheresis was 0.52F0.17 versus 0.45F0.18 in the product ( p= 0.502). 3.8. BDC can be successfully isolated from apheresis product We have optimized an isolation protocol for BDC, based on the use of the CMRF-44 monoclonal antibody and magnetic bead technology to purify BDC after overnight culture (Lopez et al., submitted for publication). Following apheresis, CMRF-44 antigen expression in vitro was not altered and purification
Fig. 7. Allogeneic MLR evaluation of enriched BDC prepared from apheresis MNC. Freshly harvested apheresis cells (fresh) were cultured overnight (cultured), labeled with CMRF-44 biotinylated antibody and enriched with a positive immunoselection column. Both positive and negative fraction cells were collected and tested. A titration of APC from each origin was cultured with isolated allogeneic T-cells, as described in Materials and methods.
of the BDC was readily achieved (Fig. 6). Furthermore, the separation of BDC from both fresh and frozen apheresis products yielded comparable BDC purity (Fig. 6). Furthermore, repeated BDC isolations (n=4) performed on the same frozen apheresis material yielded very consistent results (data not shown). The BDC purified in this way from apheresis material were fully functional in an allogeneic MLR (Fig. 7).
4. Discussion Clinical use of DC requires a highly reproducible and GMP applicable method that provides sufficient yields of nonactivated cells that are fully functional upon cryopreservation. Here, we evaluated the use of two software protocols for the collection of BDC using the Cobe Spectra apheresis machine and established that the automated version (Auto 6.1) provides a lesser volume product and reduced platelet contaminant. We also demonstrated that the apheresis product
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contains sufficient numbers of BDC in a nonactivated state suitable for immunotherapy. No skewing of subset composition was observed allowing further subset fractionation, if appropriate. The data suggests that BDC are mobilized during the procedure and although a 10-l apheresis procedure is sufficient, if necessary, a 15-l apheresis procedure will generate an improved BDC yield. Finally, we showed that BDC can be isolated from the apheresis product and that they are also fully functional after cryopreservation. A detailed functional evaluation of BDC purified after a magnetic bead separation following apheresis is presented elsewhere (Lopez et al., in preparation). The selection of an automated protocol for the harvest of BDC has various advantages. The reproducibility and the operator independence permits the standardization of the method across laboratories, a particularly relevant issue when multi-center protocols are envisaged, as will be important in evaluating DC cancer immunotherapy protocols. Apheresis using automated Cobe AutoPBSC software has also been used to obtain MNC for the preparation of MoDC and these were likewise fully functional; however, details on the apheresis procedure are not available for comparison (Lewalle et al., 2000). Apheresis MNC products have been used successfully for the production of Mo-DC from monocytes (Thurner et al., 1999; Goxe et al., 2000; Lewalle et al., 2000) and Langerhans cells from CD34+ progenitors but again, data on yields were not provided (Gatti et al., 2000). Apheresed MNC preparations have also been used for density gradient separation of BDC but no information has been published regarding the yields (Fong et al., 2001). The reduced product volume facilitates further BDC isolation procedures including the labeling with monoclonal antibodies. The Cobe AutoPBPC program provided lesser platelet contamination of the sample. This feature is particularly relevant, since platelets are known for their capacity to activate BDC; for example, high platelet contamination has been shown to hinder Mo-DC generation from apheresis material both in melanoma patients and healthy volunteers (Glaser et al., 1999). Critical to the clinical use of the pheresed material is the ability to handle the products in a closed clinical grade environment. Clinical grade Mo-DC has been prepared from pheresed MNC after processing with
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the Cobe Spectra cell separator (Rouard et al., 2000). This methodology can now be applied to BDC. The product can then be enriched significantly for BDC, whilst maintaining sterile Good Manufacturing Practice (GMP) procedures, using a BDC-specific mAb for positive cell selection. Cryopreservation of DC preparations will also be highly advantageous for clinical scheduling. Protocols examining the generation of Mo-DC have shown that they may be stored safely frozen either as PBMC or as matured Mo-DC (Thurner et al., 1999; Lewalle et al., 2000). It has also been demonstrated that antigenloaded Mo-DC obtained from apheresis protocols can be stored frozen, retaining their stimulatory function (Feuerstein et al., 2000; Lewalle et al., 2000). Our data shows that BDC can also be obtained from frozen PBMC collected by apheresis. Thawing cells prior to an overnight incubation before the antibody-mediated separation of DC makes this a very convenient protocol. Mo-DC has been generated from the apheresis products obtained from patients with multiple myeloma (Tarte et al., 1997) and chronic myeloid leukemia (Zheng et al., 2000). Indeed, we now have data that establishes the optimal BDC harvesting time in G-CSF and cyclophosphamide-treated patients with non-Hodgkin lymphoma and multiple myeloma, who are undergoing mobilization for blood stem cell collection (Vuckovic et al., in preparation). The data also predict that sufficient numbers of BDC will be obtained from apheresis in those patients without the need of additional mobilization. However, various mobilizing conditions have been used to optimize the generation of monocyte and CD34+-derived DC, resulting in changes in the phenotype and/or function of various cell types including T-lymphocytes NK and DC (Gazitt, 2000; Roth et al., 2000). Stem cell factor (SCF) mobilization in patients with breast cancer resulted in minimal increases in BDC populations (Menedez et al., 2001). Flt-3 ligand mobilizes BDL in healthy volunteers (Maraskovsky et al., 2000) and is currently being investigated by several groups for its ability to mobilize BDC in patients. DC are subject to various physiological influences that may change their status of activation and/or mobilization. We have observed that stress (surgery and extreme exercise) prompts mobilization of BDC without activation (Ho et al., 2001). Likewise, we
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have documented changes in certain disease states (Summers et al., 1999; Vuckovic et al., 1999; Brown et al., 2001). Given their responses to environmental influences, it was not inconceivable that apheresis would change BDC differentiation/activation. Our data on the evaluation of whole DC suggest that the apheresis procedure does not induce the activation state of collected BDC; a highly desirable feature allowing for in vitro optimization of BDC function for immunotherapy protocols. Differential activation of a proportion of cells within certain DC subsets (MacDonald et al., in press) may escape detection and will be the subject of future analyses. In contrast, maturation of Mo-DC from an apheresis product was reported to occur earlier than for cells drawn from fresh blood samples (Thurner et al., 1999). We have shown here that BDC isolated from apheresis MNC preparations exhibited similar functions to BDC drawn by direct venipuncture. In conclusion, this study describes critical information required to generate sufficient, nonactivated and fully functional BDC in a standard manner for DC-based cancer immunotherapy studies.
Acknowledgements We gratefully acknowledge the enthusiastic participation of the volunteers recruited for this study; we thank Drs. Cameron Turtle, Chris Ho, Geoff Hill and Nick Murray for constructive discussions and Gilles Bioley for help with the functional evaluation of apheresis products. We acknowledge the support Miltenyi Biotech (Bergisch Gladbach, Germany) for immuno-isolation reagents. This work was funded by the Mater Medical Research Institute and by GAMBRO BCT (Lakewood CO).
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