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Selective in Vivo Mobilization with Granulocyte Macrophage Colony-stimulating Factor (GM-CSF)/Granulocyte-CSF ascompared to G-CSF Alone of Dendritic Cell Progenitors from Peripheral Blood Progenitor Cells in Patients with Advanced Breast Cancer Undergoing Autologous Transplantation¹

Division of Hematology/Oncology, Beth Israel Deaconess Medical Center, Boston, Massachusetts 02215 [D. A., R. J., J. L., J. M., L. K.], and Cancer Pharmacology, Dana-Farber Cancer Institute, Boston, Massachusetts 02115 [Z. W., J. G., A. E., P. R., K. A., D. K.]

	ABSTRACT

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Dendritic cells (DCs) are potent antigen-presenting cells that are essential for the initiation of T cell-mediated immunity. DCs develop from myeloid progenitor populations under the influence of granulocyte macrophage colony-stimulating factor (GM-CSF) and pass through an intermediate stage of maturation that is characterized by CD14 expression. Interest has focused on generating human-derived DCs for antigen-specific tumor vaccines to be used as adjuvant immunotherapy in minimal disease settings, such as after autologous transplantation. In the present study, mobilized peripheral blood progenitor cells (PBPCs) were obtained from 18 patients with locally advanced or metastatic breast cancer preparing to undergo autologous stem cell transplantation. PBPCs mobilized in 10 patients with GM-CSF for 1 week, followed by the combination of GM-CSF and G-CSF, were compared with those obtained from patients receiving G-CSF alone with respect to the presence of DC progenitors and the capacity to generate functionally active mature DCs. PBPCs mobilized with GM-CSF/G-CSF were markedly enriched for CD14+ DC progenitor cells as compared with those mobilized with G-CSF alone. Consistent with an immature progenitor population, the CD14+ cells express Ki-67 antigen but not nonspecific esterase. CD14+ cells purified by fluorescence-activated cell sorting from PBPCs mobilized with either regimen and cultured for 1 week in GM-CSF and interleukin-4 generated nearly pure populations of cells with characteristic DC phenotype and function. The addition of GM-CSF to the mobilization regimen resulted in greater yields of functionally active DCs for potential use in posttransplant immunotherapy.

	INTRODUCTION

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DCs³ are bone marrow-derived leukocytes that excel in antigen presentation and the initiation of T cell-mediated immunity (1) . DCs express costimulatory molecules [such as CD80 (B7-1), CD86 (B7-2), ICAM-1, and LFA-3] that are essential for activation of primary cellular immune responses (2 , 3) . Tumor cells that present antigens in the absence of costimulation induce loss of immune recognition (4, 5, 6) . By contrast, anergy is reversed through immunization with DCs manipulated to express tumor-specific antigens. Recent studies in animal models have shown that DCs pulsed with tumor peptides or fused with malignant cells are effective in protecting animals from tumor challenge and mediate tumor regression in cancer-bearing hosts (7, 8, 9) . Moreover, a clinical study using idiotype-pulsed DCs for the treatment of B-cell lymphoma has demonstrated efficacy without significant morbidity (10) . The development of DC vaccines for immunization in minimal disease settings, such as after autologous transplantation, represents another focus for investigation.

DCs develop from myeloid progenitor populations under the influence of cytokines, most notably GM-CSF (11, 12, 13, 14, 15, 16, 17) . Previous studies have shown that DC precursors isolated from murine peripheral blood and bone marrow produce DC progeny when exposed to GM-CSF (18) . Human CD34+ cells derived from bone marrow, cord blood, or PBPCs generate significant numbers of DCs when cultured in the presence of GM-CSF, TNF, and/or IL-4 (19, 20, 21) . Mature DCs can also be generated from partially differentiated progenitor populations. In this context, significant yields of peripheral blood-derived DCs have been obtained by culturing peripheral blood precursor populations in GM-CSF and IL-4 (22) . As DCs mature from early precursor populations they pass through intermediate stages of development defined by distinctive functional and phenotypic characteristics. Immature cells exemplified by LCs of the skin are capable of antigen uptake, but demonstrate poor stimulatory capacity of T cells (23) . LCs characteristically express CD1a, E-cadherin, and Lag antigen and contain cytoplasmic inclusion bodies known as Birbeck granules (15) . On culture with GM-CSF, these cells develop a mature phenotype associated with diminished ability to process exogenous antigen. In addition, the up-regulated expression of costimulatory and adhesion molecules on mature DCs is associated with increased activity in allogeneic MLRs. DCs may also be derived from precursor cells related to the monocytic lineage. This category of intermediately differentiated DCs express the CD14 antigen (19 , 24) . These cells are distinguished from mature monocytes by faint expression of nonspecific esterase and by a proliferative capacity, as documented by expression of Ki-67 antigen.

A potential clinical source of DC precursors is PBPCs mobilized with cytokines from patients before undergoing high-dose chemotherapy with stem cell rescue. Treatment with myeloid CSFs results in a 20- to 40-fold increase in circulating colony-forming unit-GM per kilogram (25) . In the present study, we show that PBPCs mobilized in vivo with GM-CSF/G-CSF are enriched for CD14+ DC progenitors as compared with those mobilized with G-CSF alone. When cultured in vitro with GM-CSF and IL-4, CD14+ progenitors produced a nearly pure population of mature DCs with a characteristic phenotype. These results demonstrate that DC progenitors can be selectively mobilized with GM-CSF/G-CSF in vivo.

	PATIENTS AND METHODS

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Patient Population.
Samples were obtained from 18 women with locally advanced or metastatic breast cancer. Patients were treated with induction chemotherapy consisting of paclitaxel, doxorubicin, or cyclophosphamide and then enrolled in an Institutional Review Board approved protocol for high-dose chemotherapy with stem cell rescue. Fifteen of 18 patients received cyclophosphamide as the chemotherapy component of their mobilization regimen, 2 patients were treated with a taxane, and 1 patient received no chemotherapy. All patients subsequently received daily injections of hematopoietic growth factor(s) until the completion of stem cell collections. Ten patients received daily injections of 5 µg/kg GM-CSF for 1 week, followed by the combination of GM-CSF and G-CSF each at 5 µg/kg, whereas eight patients received daily injections of G-CSF alone at 5 µg/kg until completing stem cell collections. Patients began stem cell collections once their WBC was 3000 cells/ml, typically 10 days after the initiation of cytokine therapy. The patients then underwent serial leukaphereses to collect PBPCs for rescue after high-dose chemotherapy. These collections (1 ml) were obtained for the purposes of this study.

Sorting of PBPCs before in Vitro Culture.
PBPCs were suspended in RPMI 1640 containing 10% heat-inactivated human AB serum (Sigma Chemical Co., St. Louis, MO), 2 mM L-glutamine, 100 units/ml penicillin, and 100 µg/ml streptomycin for 1 h at 37°C. Nonadherent cells containing primarily T cells, B cells, and granulocytes were removed, and the adherent cells were incubated overnight at 37°C. After 12–16 h, the culture was washed gently with media and the loosely adherent cells were harvested leaving firmly adherent monocytes. An aliquot was removed for FACS analysis and MLR studies. A fraction of the loosely adherent PBPCs was placed in 6-well plates (1 x 10⁶ cells/ml) in medium containing 1000 units/ml GM-CSF (Immunex, Seattle, WA) and 1000 units/ml IL-4 (Genzyme, Boston, MA). The remaining cells were incubated with primary murine antihuman CD14 antibody (Coulter, Miami, FL) for 30 min on ice. After two washes, the cells were then incubated with a secondary goat antimurine antibody conjugated with FITC or PE (Sigma Chemical Co.) for indirect fluorescence labeling. The cells were subjected to FACS sorting and segregated into CD14-positive and -negative populations. The sorted populations were then cultured in GM-CSF and IL-4 for 1 week.

Preparation of Peripheral Blood Monocytes and T Cells.
Peripheral blood mononuclear cells were derived from leukopaks obtained from healthy donors that were subjected to Ficoll-Hypaque (Pharmacia, Piscataway, NJ) density gradient centrifugation (d = 1.077, 400 x g). Cells were suspended in RPMI 1640 containing 10% heat-inactivated human AB serum, 2 mM L-glutamine, 100 units/ml penicillin, and 100 µg/ml streptomycin for 1 h. Nonadherent cells were harvested, and T cells from that fraction were purified using a T-cell column or nylon wool (R&D Systems, Minneapolis, MN). The adherent population was incubated for 12–16 h, and the loosely adherent cells were removed by gentle washing of the culture flask. The strongly adherent cells represented a mature monocyte population and were maintained in media with human sera in the absence of cytokines.

Monoclonal Antibodies and FACS Analysis.
Phenotypic characterization of the cell populations before, as well as after, in vitro culture was performed using monoclonal antibodies specifically reactive with the cell surface antigens CD14, CD80, CD86, CD83, CD54, and HLA-DR (Coulter, Miami, FL). Cells were incubated with primary murine antihuman antibody for 30 min on ice. After two washes, the cells were then incubated with a secondary goat antimurine antibody conjugated with FITC or PE (Sigma Chemical Co.) for indirect fluorescence labeling. Alternatively, cells were incubated with PE-conjugated anti-CD83 antibody and a nonspecific murine antibody as a control. Cells were then washed, fixed with 2% paraformaldehyde, and evaluated by flow cytometric analysis.

	Immunocytochemistry Staining

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Analysis of DR Expression.
Cells were spun onto glass slides using a cytocentrifuge. The cell preparations were fixed in acetone for 5 min and treated with 100 µg/ml avidin/1% normal horse serum in PBS (pH 7.2) for 30 min at 4°C to block endogenous avidin-binding activity. The cells were then incubated for 1 h at room temperature with 10 µg/ml murine antihuman DR antigen (Coulter). Hydrogen peroxide solution (0.3%) containing 20 µg/ml biotin was used to quench endogenous peroxidase activity for 30 min at 4°C. The sample was incubated with 1:100 biotinylated F(ab')2 fragment of horse antimouse IgG (Vector Laboratories, Burlingame, CA) for 45 min at room temperature, washed with PBS, and incubated for 30 min at room temperature with ABC reagent solution (Vector Laboratories). Specific binding was detected with 3-amino-9-ethyl carbazole solution (Vector Laboratories). Slides were counterstained with Mayer’s hematoxylin solution.

Nonspecific Esterase Staining.
Cytospin cell preparations were fixed in citrate-acetone, dried, and incubated with ethylene glycol-naphthyl acetate solution in Trizmal buffer for 20 min at 37°C protected from light. Cells were counterstained for 5 min in Mayer’s hematoxylin solution.

Analysis of Ki-67 Expression.
Cytospin cell preparations were treated with heated citrate buffer and incubated with a murine antihuman Ki-67 antibody, as described for an automated Ventana procedure (Immunotech, Marseille, France).

MLR Studies.
Allogeneic T cells (1 x 10⁵) were plated in 96-well round bottom plates in a final volume of 200 µl of RPMI 1640 with 10% human serum. APCs were seeded at 3 x 10³, 1 x 10³, 3 x 10², and 1 x 10² cells per well. T cells and antigen-presenting cells (APCs) were cocultured for 5 days. T-cell proliferation was determined by measuring thymidine incorporation after the addition of 0.5 µCi/well of ³H-TdR for 12–16 h. The data are reported as the mean cpm of triplicate wells.

	RESULTS

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Characterization of PBPCs Mobilized in Vivo with GM-CSF and G-CSF as Compared to G-CSF Alone.
PBPCs obtained from 10 patients with advanced breast cancer undergoing stem cell mobilization with GM-CSF and G-CSF were compared with those from 8 patients whose stem cells were mobilized with G-CSF alone. Loosely adherent cells were separated from nonadherent B cells, T cells, and granulocytes and the firmly adherent mature monocytes, and were then subjected to FACS analysis. The mean total CD34+ cells/kg was not statistically different in the PBPC collections mobilized with GM-CSF/G-CSF as compared with those obtained with G-CSF (4.0 versus 2.3, P = 0.22; Table 1 ). By contrast, the population of CD14+ cells was significantly increased in PBPCs mobilized with GM-CSF/G-CSF (range, 17–74%; mean, 51%) as compared with those mobilized with G-CSF alone (range, 1–20%; mean, 8%; P < 0.0001). DR, CD80, CD54, and CD86 expression was also more pronounced in PBPCs mobilized with GM-CSF/G-CSF, whereas CD83 was absent in both populations (Fig. 1) . CD14+ cells obtained after either mobilization failed to show evidence of nonspecific esterase activity. Immunocytochemical staining of the CD14+ cells for Ki-67 was uniformly strongly positive, whereas adherent monocytes showed no evidence of Ki-67 expression (Fig. 2) .

View larger version (21K): [in this window] [in a new window] [Download PPT slide]   Fig. 1. Phenotypic profile of PBPCs generated from patients mobilized with GM-CSF and G-CSF as compared with G-CSF alone. PBPCs were mobilized in vivo with chemotherapy, followed by G-CSF alone versus GM-CSF for 1 week, followed by the combination of GM-CSF and G-CSF. After collection, PBPCs were plated for 1 h and the nonadherent fraction was removed. The adherent population was incubated overnight in human sera. Loosely adherent cells were subsequently harvested, and an aliquot was taken for FACS analysis. The cells were then incubated with murine antibodies against the indicated antigens. After two washes, the cells were incubated with goat antimouse immunoglobulin conjugated to FITC and then fixed in paraformaldehyde. Histograms were generated by cytometric analysis.

View larger version (75K): [in this window] [in a new window] [Download PPT slide]   Fig. 2. Phenotypic characterization of CD14+ sorted PBPCs. A, nonspecific esterase stain. CD14+ cells were separated from nonadherent PBPCs using FACS and spun onto glass slides. Cytospins were fixed in citrate-acetone, air dried, incubated with ethylene glycol-naphthyl acetate solution, and counterstained in Mayer’s hematoxylin. B, positive control. Peripheral blood containing monocytes and mature T cells were subjected to nonspecific esterase staining as a positive control. C, Ki-67 antigen. CD14+ PBPCs were spun onto glass slides, treated with heated citrate buffer, and incubated with murine antihuman Ki-67 antibody. Purified monocytes used as a negative control failed to demonstrate reactivity with anti-Ki-67.

View larger version (40K): [in this window] [in a new window] [Download PPT slide]   Fig. 3. Phenotypic profile of unselected PBPCs, CD14+ PBPCs, and CD14- PBPCs after 1 week of culture in GM-CSF and IL-4. PBPCs were mobilized in vivo with chemotherapy, followed by GM-CSF/G-CSF. After collection, PBPCs were plated for 1 h in human sera, and nonadherent cells were removed. The cells were then cultured overnight. The loosely adherent cells were then harvested, and an aliquot of the cells was incubated with murine antihuman CD14. After washing and incubation with goat antimouse immunoglobulin conjugated to FITC, the cells were segregated by FACS into CD14+ and CD14- populations. The unselected, CD14+ and CD14- fractions were cultured with GM-CSF/IL-4 for 1 week and then incubated with the indicated primary murine antihuman antibodies and goat antimouse immunoglobulin conjugated to FITC or PE-conjugated anti-CD83 antibody and nonspecific murine antibody as a control. The cells were fixed in paraformaldehyde, and histograms were generated by cytometric analysis.

View larger version (97K): [in this window] [in a new window] [Download PPT slide]   Fig. 4. MHC class II staining of unselected PBPCs after 1 week of culture in GM-CSF and IL-4. PBPCs were harvested after 1 week of culture in GM-CSF and IL-4. Cytospins were prepared and fixed in acetone. The cells were then incubated for 1 h at room temperature with mouse antihuman DR antigen, followed by biotinylated F(ab')2 fragment of horse antimouse IgG. Slides were counterstained with hematoxylin solution. A, a mixed population of brightly stained DCs adjacent to more weakly staining monocytes (x40 magnification). B, two brightly stained DCs with a weakly stained monocyte in the periphery of the field (x100 magnification).

View larger version (38K): [in this window] [in a new window] [Download PPT slide]   Fig. 5. Allogeneic MLR studies with DCs generated from CD14+ and CD14- PBPCs. PBPCs mobilized with GM-CSF/G-CSF or G-CSF alone were segregated into CD14+ and CD14- fractions. The cells were cultured for 1 week with GM-CSF and IL-4. T cells were plated at 1 x 105 cells/well and cocultured with DCs generated from G-CSF-mobilized CD14+ cells (), DCs generated from G-CSF-mobilized CD14-cells (), DCs generated from GM-CSF-mobilized CD14+ cells (), and DCs generated from GM-CSF-mobilized CD14- cells () at a T cell:APC ratio of 30:1. The results (mean ± SD) are representative of three independent experiments, each performed in triplicate.

	DISCUSSION

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Cytokine-mobilized PBPCs are rich in myeloid progenitor populations (25) . Several studies have demonstrated that CD34+ cells derived from G-CSF mobilized PBPCs and cultured with GM-CSF and TNF generate mixed populations of cells that include large numbers of functionally active LCs and mature DCs (21) . G-CSF-mobilized PBPCs depleted of CD34+ cells also produce significant yields of DCs when cultured in GM-CSF and IL-4 (27) . However, little is known about whether PBPCs mobilized with GM-CSF are enriched for the presence of partially differentiated DC precursors. The present study demonstrates that the addition of GM-CSF to the mobilization regimen markedly increases the number of nonadherent CD14+ DC progenitors in the stem cell product, as compared with that obtained with G-CSF alone. Unlike mature monocytes, these cells do not express nonspecific esterase. Moreover, nuclear staining for Ki-67 demonstrates that the CD14+ cells represent a precursor population with proliferative capacity. These cells exhibit moderate class II expression, whereas costimulatory molecules CD80 and CD86 were largely absent and CD83 was undetectable. When cultured for 1 week in vitro with GM-CSF and IL-4, CD14+ PBPCs generated a nearly pure population of cells with a characteristic DC phenotype, as demonstrated by FACS analysis. These cells expressed the relatively specific DC marker CD83 and potently stimulated allogeneic T-cell proliferation. CD14- PBPCs represented a greater percentage of cells mobilized with G-CSF alone. CD14- PBPCs cultured for 1 week in GM-CSF and IL-4 generated cells that did not express CD83 and were less active in MLR studies. The final yield of DCs as a percentage of the starting PBPC population was significantly greater for patients mobilized with GM-CSF/G-CSF. Therefore, although CD14+ DC precursors were present in PBPCs mobilized with either GM-CSF/G-CSF or G-CSF alone, the use of GM-CSF was associated with significantly higher yields of these DC progenitors and, as such, resulted in an increase in mature DCs generated in vitro.

Previous studies have demonstrated that DCs produced from CD34+ PBPCs from cancer patients are potent stimulators of allogeneic MLR (28) . In another study, DCs derived from PBPCs mobilized from patients with multiple myeloma were shown to be functionally active (27) . In the present study, DCs generated from heavily pretreated patients with advanced breast cancer undergoing stem cell mobilization were also found to be functionally active. Thus, the postautologous transplant period may be an ideal setting for the effective use of antitumor DC-based vaccines. Patients treated with high-dose chemotherapy are often able to achieve a complete remission, but subsequently relapse. These findings have suggested that patients achieving a minimal disease state retain tumor clones that are chemotherapy resistant. These residual tumor cells may, however, be susceptible to active specific immunotherapy with DC-based vaccines. Our study is the first to demonstrate that chemotherapy followed by the combination of GM-CSF and G-CSF may selectively mobilize DC progenitors in vivo in the stem cell product and that these cells can generate relatively pure populations of functionally active mature DCs. This approach to stem cell mobilization, therefore, results in an excellent source of DC progenitors that could be used for immunotherapeutic strategies in the posttransplant period.

	ACKNOWLEDGMENTS

 
We thank Dr. Lynne Uhl and the nursing staff of the leukapheresis unit at the Beth Israel Deaconess Medical Center for help in acquiring samples and Nancy Giallombardo, Courtney Price, and Tracy Dane for assistance in the preparation of the manuscript.

	FOOTNOTES

 
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

¹ Sponsored in part by a grant from the Immunex Corporation.

² To whom requests for reprints should be addressed, at 330 Brookline Avenue, Boston, MA 02215. Phone: (617) 667-9920; Fax: (617) 667-9922; E-mail: davigan{at}bidmc.harvard.edu

³ The abbreviations used are: DC, dendritic cell; GM-CSF, granulocyte macrophage colony-stimulating factor; TNF, tumor necrosis factor; IL, interleukin; LC, Langerhans’ cell; PBPC, peripheral blood progenitor cell; MLR, mixed lymphocyte reaction; FACS, fluorescence-activated cell-sorting; APC, antigen-presenting cell.

Received 3/31/99; revised 7/27/00; accepted 8/ 2/99.

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