Progenitor Cell Mobilisation

Etoposide (VP-16) plus G-CSF mobilizes different dendritic cell subsets than does G-CSF alone

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Dendritic cells (DCs) are antigen-presenting cells that are critical to the generation of immunologic tumor responses. Myeloid DCs (DC1) express myeloid antigen CD11c; lymphoid DCs (DC2) express CD123+ and are CD11c. Analysis of DC subsets from peripheral blood progenitor cells (PBPC) collected from normal donors mobilized with G-CSF shows a predominance of DC2 cells. Whether PBPCs mobilization by chemotherapy yields different subsets of DCs has not been studied. We analyzed DC subsets in apheresis products from 44 patients undergoing autologous stem cell transplantation from 6/00 to 5/01. Patients received either G-CSF alone (10 μg/kg per day, n=11) or etoposide (2 g/m2) plus G-CSF (n=33) for progenitor cell mobilization. The patients were apheresed for 2–10 days (median 3) to reach a minimum of 2.0×106 CD34+ cells/kg. Patients receiving G-CSF alone mobilized significantly more total DC2s than did those receiving etoposide plus G-CSF (median 6.2×106/kg vs 2.9×106/kg, P=0.001). The DC2/DC1 ratio was also significantly different in the two groups, with the G-CSF group having a higher ratio (median 1.2 vs 0.4, P<0.001). We conclude that the combination of chemotherapy plus G-CSF yields different mobilized dendritic cell subsets than does G-CSF alone.


Dendritic cells (DCs) are antigen-presenting cells and the only cells that can prime naive T cells to a new antigen.1 Clinical studies have been reported using DCs as immunotherapeutic vaccines for patients with multiple myeloma, prostate cancer, and other diseases with encouraging early results.2,3,4 DCs have two distinct lineages: myeloid DCs (DC1) and lymphoid DCs (DC2). DC1s express myeloid antigen CD11c+; produce a high level of interleukin 12 when stimulated with tumor necrosis factor, and drive T-cell differentiation into Th1. DC2s are CD123+ and CD11c; after appropriate activation, DC2s induce T-cell differentiation into Th2 cells.

Recipients of allogeneic BMT receiving larger numbers of DC2 cells in the allograft have been reported to have a lower incidence of chronic graft-versus-host disease (GVHD) and increased incidence of disease relapse.5 Whether the total number of DC2s, or specific DC subsets, infused after autologous stem cell transplantation affect survival is not yet studied. As a preliminary analysis, we prospectively examined the total number of DCs mobilized, and the DC2/DC1 ratio, in 44 consecutive patients undergoing autologous stem cell transplant at the Cleveland Clinic Foundation from 6/00 to 5/01.

Patient and methods

A total of 44 consecutive patients undergoing autologous stem cell transplant from 6/00 to 5/01 at the Cleveland Clinic Foundation were studied. Clinical characteristics of the patients are shown in Table 1. All patients were treated on protocols approved by the Institutional Review Board of the Cleveland Clinic Foundation and gave written informed consent.

Table 1 Patient population

PBPC mobilizing regimen

All patients received one of the two mobilizing regimens. In all, 11 patients received G-CSF 10 μg/kg/day subcutaneously, with leukapheresis beginning on the fifth day of G-CSF administration. A total of 33 patients received etoposide (2 g/m2) plus G-CSF (10 μg/kg subcutaneously daily) with leukapheresis beginning once WBC recovery was greater than 1000/mm3. This was a nonrandomized study; a higher percentage of patients with non-Hodgkin's lymphoma (NHL) received etoposide plus G-CSF largely because a significant percentage of these patients had actively growing disease and the combination of etoposide plus G-CSF was used to secure disease control pretransplant.

PBPC collection and processing

PBPC collection was performed on a COBE Spectra leukapheresis machine (COBE, Denver, CO, USA). Patients were apheresed for at least 2 days, with a minimum yield of 2.0×106 CD34+ cells/kg. Apheresis continued for 5 days, or when 7.0×106 CD34+ cells/kg were collected, whichever came first.

Dendritic cell determination by flow cytometry

Fresh peripheral blood progenitor cell products were processed for flow cytometry to quantitate myeloid (CD11c+) and lymphoid (CD123+) subclasses of peripheral blood DCs. Since neither CD123 nor CD11c are specific markers of DCs, a four-color flow cytometry assay was used with a gating strategy that employed a cocktail of antibodies to first separate neutrophils, monocytes, lymphocytes, and NK cells from basophils and DCs. Basophils were then distinguished from DCs based on the levels of HLA-DR expression: basophils lack HLA-DR expression, whereas this antigen is highly expressed on DCs. This assay utilized commercially available antibodies in a procedure optimized for flow cytometry by the manufacturer.6

Briefly, 1×106 cells were simultaneously stained using a lineage cocktail (lin) of FITC-labelled monoclonal antibodies against T cells (CD3), monocytes (CD14), neutrophils (CD16), B cells (CD19, CD20), and natural killer cells (CD16, CD56); PE-labelled CD123 (anti-IL-3R alpha); PerCP-labelled anti-HLA-DR; and APC-labelled CD11c. Negative controls for nonspecific antibody staining included PE-labelled mouse IgG1, PerCP-labelled mouse IgG2a, and APC labelled mouse IgG2a. After incubation in the dark for 15 min at room temperature, the cells were vortexed and washed in PBS. The cells were then centrifuged for 5 min at 300×g, and the supernatant discarded. The cell pellet was resuspended in 0.3 ml of 0.5% paraformaldehyde. After diluting the sample with PBS, pH 7.2, with a flow rate set to low, 100 000 ungated events (excluding debris) were acquired to a list mode file according to light scatter properties using a FACSCaliburTM flow cytometer and CellQuestTM software (Becton-Dickenson, San Jose, CA, USA). The list mode file was analyzed sequentially in the following steps. First, using an ungated forward vs side scatter dot plot, a gate (R1) was adjusted to exclude debris and dead cells. Next, an anti-HLA vs lineage cocktail plot was constructed to select lineage dim/negative events within this region (R2). Within the HLA-DR/lindim/negative events, a gate (R3) was selected to include anti-HLA-DR vs CD123 or anti-HLA-DR vs CD11c dot plots. Using these plots the following cell populations were individually quantitated: basophils (anti-HLA-DR /CD123+), CD123+ dendritic cells (anti-HLA-DR+ and CD123+) and CD11c+ dendritic cells (anti-HLA-DR+/CD11c+). Results were reported as a percentage of the total events (excluding debris). From the data, a ratio of lymphoid DC2 (HLA-DR+/linnegative/CD11c/CD123+) to myeloid DC1 (HLA-DR+/linnegative/CD11c+/CD123) dendritic cells was determined. The absolute numbers of the dendritic cell subsets were calculated by multiplying the percentage of HLA-DR+/CD123+ or HLA-DR+/CD11c+ dendritic cells derived above by the absolute leukocyte count obtained using a Sysmex SE9500 (TOA Medical Electronics, Japan) cell counter. For the purposes of calculating the dose of cells collected in each product, the absolute number of DC1 and DC2 were expressed as the number of cells (×106)/kg of patient weight.

Statistical analysis

Categorical variables were summarized as frequencies and percentages and compared between the two mobilizing regimens using the χ2 test. Continuous variables were summarized as the median and range and compared between mobilizing regimens using the Wilcoxon rank sum test. Multiple linear regression analysis was used to compare DCs between the mobilizing regimens, adjusting for the variables that differed between regimens. Cox proportional hazards analysis was used to assess the correlation between DCs and survival. Analyses were conducted using SAS® software. All statistical tests were two-sided, and P<0.05 was used to indicate statistical significance.


Mobilization with G-CSF was well tolerated. Mobilization with etoposide plus G-CSF resulted in significant neutropenia beginning at a median of seven days after the infusion of etoposide and lasting for an average of 5 days. Leukapheresis began at a median of 14 days after etoposide administration.

The total CD34+ yield was significantly higher in the etoposide +G-CSF group, as shown in Table2. The patients receiving etoposide +G-CSF required fewer days of leukapheresis.

Table 2 CD34+ cell yield

The two mobilizing regimens yielded different results with respect to dendritic cell mobilization, as shown in Table3. Patients receiving G-CSF alone mobilized a significantly higher number of total DC2 cells, as well as a higher DC2/DC1 ratio.

Table 3 Dendritic cell mobilization

Since patients receiving etoposide+G-CSF tended to have more robust CD34+ cell mobilization, they generally required fewer aphereses to collect the target dose of CD34+ cells. We questioned whether the fact that patients receiving etoposide had fewer total days of collection might influence the DC yield. Since all patients required a minimum of 2 days of apheresis, we could compare the DC yield and the DC2/DC1 ratio between the two regimens separately for days 1 and 2 of apheresis. Results were consistent with those from total days of apheresis, and are shown graphically in Figures 1a–c.

Figure 1

Box and whiskers plot of DC2/DC1 ratio according to mobilizing regimen: (a) day 1 of pheresis, (b) day 2 of pheresis, (c) all days of pheresis. The box represents the middle 50% of the data; whiskers represent the range. The thick line represents the median. Individual data points are shown as open circles.

We also considered the possibility that the differences in DC mobilization were related to CD34+ cell yield. We were also concerned about differences in age, diagnosis, and days of apheresis between the mobilizing regimens. However, when we conducted a multivariable analysis to adjust for these differences, we still found that G-CSF resulted in more DC2s and a higher DC2/DC1 ratio than did etoposide+G-CSF (P<0.001).

The median follow-up of survivors is 12 months. With limited follow-up, DC mobilization has not influenced survival.


Dendritic cells are critical to immunologic tumor responses. DCs bind and present tumor antigens to T lymphocytes in a way that is immunogenic. Dendritic cells may play a role in the graft-versus-tumor effect. It has been shown that G-CSF mobilizes peripheral blood progenitor cells that contain higher doses of DC2 than do marrow transplants.7 It has further been hypothesized that G-CSF stimulated PBPCs do not result in overwhelming GVHD because of the fact that the graft contains predominantly DC2 dendritic cells.7 Additionally, it has been reported that recipients of T cell nondepleted allogeneic bone marrow containing a large number of DC2 cells have a lower incidence of GVHD and an increased incidence of leukemic relapse, strongly suggesting that the DC2 content was associated with both GVHD and the graft-versus-leukemia effect.5 Thus, the study of DC content in mobilized PBPC products has potential clinical importance.

Mobilizing dendritic cells as immunotherapy has been described in patients with metastatic colon cancer, and dendritic cells have also been used as an immunologic adjuvant treatment of hormone refractory prostate cancer and multiple myeloma.2,3,4 The role of dendritic cells and dendritic cell subtypes in autologous transplantation is yet undefined. However, it would be of interest if different mobilizing regimens might yield different subsets of dendritic cells as a platform to potentially utilize DCs immunotherapeutically. One study has suggested that different mobilizing regimens yield similar DC subsets.8 Our report, in contrast, shows that different mobilizing regimens do, in fact, yield different subsets of DCs. Specifically, we have confirmed that G-CSF-mobilized PBPCs contain predominantly DC2 cells, whereas the use of etoposide+G-CSF for PBPC mobilization yields an increase in DC1 cells and a decrease in the DC2/DC1 ratio. While it is known that different cytokines, including Flt3 ligand, stimulate DCs,9,10 our study has demonstrated that chemotherapy mobilizing regimens have the capacity to mobilize different DC subsets.

A major limitation of our study is the fact that it is retrospective. The patients were not prospectively assigned one of the two mobilizing regimens; rather it was at the choice of the treating clinician. This limits the strength of the presented data. Nevertheless, at a time when cellular engineering of hematopoietic progenitors is an important clinical research topic, simply describing the fact that different mobilizing regimens yield different dendritic cell subsets is noteworthy.

Autologous PBPC transplantation is potentially curative for many disorders, including lymphomas, that would otherwise be incurable with conventional chemotherapy. The biggest challenge, however, remains disease relapse post-transplant. In addition to more appropriate patient selection, it is likely that immunologic therapy will be needed to further reduce the risk of disease relapse after autologous BMT. DCs may be an important component of such immunotherapy. The ability to mobilize different DC subsets may be an important first step for future immunotherapy research utilizing DCs with ABMT.


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Bolwell, B., Sobecks, R., Pohlman, B. et al. Etoposide (VP-16) plus G-CSF mobilizes different dendritic cell subsets than does G-CSF alone. Bone Marrow Transplant 31, 95–98 (2003) doi:10.1038/sj.bmt.1703791

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  • dendritic cells
  • progenitor cell mobilization
  • autologous bone marrow transplant

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