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© 2000 American Society for Clinical Oncology Preoperative Mobilization of Circulating Dendritic Cells by Flt3 Ligand Administration to Patients With Metastatic Colon CancerFrom the Departments of Medicine, Surgery, Immunology, and Pathology, Duke University Medical Center, Durham, NC, and Immunex, Corporation, Seattle, WA. Address reprint requests to Michael A. Morse, MD, Duke University Medical Center, Box 2606, Durham, NC 27710.
PURPOSE: To evaluate preoperative dendritic cell (DC) mobilization and tumor infiltration after administration of Flt3 ligand (Flt3L) to patients with metastatic colon cancer. PATIENTS AND METHODS: Twelve patients with colon cancer metastatic to the liver or lung received Flt3L (20 µg/kg/d subcutaneously for 14 days for one to three cycles at monthly intervals) before attempted metastasectomy. The number and phenotype of DCs mobilized into peripheral-blood mononuclear cells (PBMCs) were evaluated by flow cytometry. After surgical resection, metastatic tumor tissue was evaluated for DC infiltration. In vivo immune responses to recall antigens were measured. RESULTS: After Flt3L administration, on average, the total number of leukocytes in the peripheral blood increased from 5.9 ± 1.0 x 103/mm3 to 11.2 ± 3.8 x 103/mm3 (mean ± SD, P = .0001). The percentage of CD11c+CD14- DCs in PBMCs increased from 2.4% ± 1.8% to 8.8% ± 4.7% (P = .004). Delayed-type hypersensitivity (DTH) responses to recall antigens (Candida, mumps, and tetanus) showed marginally significant increases in reactivity after Flt3L administration (P = .06, P = .03, and P = .08, respectively). An increase in the number of DCs was observed at the periphery of the tumors of patients who received Flt3L compared with those of patients who had not. CONCLUSION: Flt3L is capable of mobilizing DCs into the peripheral blood of patients with metastatic colon cancer and may be associated with increases in DC infiltration in the peritumoral regions. Flt3L mobilization is associated with a trend toward increased DTH responses to recall antigens in vivo. The use of Flt3L to increase circulating DCs for cancer immunotherapy should be considered.
THE APPLICATION OF active immunotherapy to the treatment of cancer, including metastatic gastrointestinal malignancies, has been approached by a number of strategies, including administration of cytokines as well as immunizations with vaccines based on tumor cells, tumor antigens, and dendritic cells (DCs).1 Recently, Flt3 ligand (Flt3L), a cytokine with stimulatory effects on bone marrow progenitors,2 was noted to have an antitumor effect in murine models.3-6 After Flt3L administration to mice, DCs and T cells have been found in increased numbers at tumor sites4,7 and tissues such as the liver, spleen, lymph nodes, and peripheral blood.8-10 Natural killer (NK) cells may also play a role in the antitumor immune responses because they are increased in the bone marrow, thymus, peripheral blood, liver, and spleen of mice,11 and the antitumor effect of Flt3L is reduced by NK depletion.7,12 Multiple subpopulations of DCs could be identified in mice based on expression of CD11c and CD11b.8 These distinct subsets have different stimulatory effects on helper 1 T cells and helper 2 T cells with potential importance for immunotherapy.13 Although it is not known whether the immune response stimulated by Flt3L alone is antigen-specific, Flt3L can enhance antigen-specific T-cell responses to systemically administered proteins.14 Flt3L was initially administered to healthy human volunteers at doses of 10 to 100 µg/kg/d for 14 consecutive days with few side effects.15,16 The WBC count, and in particular the monocyte fraction of circulating leukocytes, increased after Flt3L administration. The number of DCs, described as lineage-negative, CD11c+ cells, increased 30-fold in the peripheral blood.16 More recent studies in healthy volunteers have demonstrated the mobilization of two populations of DCs, both the CD11c+CD14-interleukin (IL)3-receptor (R)dim myeloid DCs (also called DC1) and the CD11c-CD14-IL-3Rbright plasmacytoid DCs (also called DC2).17,18 Flt3L has also been administered concomitantly with granulocyte colony-stimulating factor (G-CSF) or granulocyte-macrophage colony-stimulating factor (GM-CSF) to patients with metastatic breast cancer, and similar increases in the number of circulating DCs were observed.19 Based on the potential of peripheral-blood DC mobilization after Flt3L administration, we hypothesized that Flt3L administered preoperatively to cancer patients before resection would increase the number of DCs in peripheral blood as well as infiltrate their tumors. We performed a pilot study of preoperative Flt3L mobilization in patients with metastatic colon cancer. Preoperative Flt3L mobilization was well tolerated, and we observed an increase in DCs in peripheral blood and a relative increase in number in peritumoral tissue. In addition, in vivo immune responses, demonstrated by skin test reactivity to recall antigens, were augmented after Flt3L.
Patients Patients were recruited from the medical and surgical oncology clinics of Duke University Medical Center from April 1998 to January 1999. For inclusion, patients were required to have metastatic colon cancer (involving the liver or lung) with a plan for an elective curative resection of the metastasis within 3 months. Exclusion criteria were chemotherapy, radiation therapy, or immunotherapy within the prior 4 weeks, history of autoimmune disease including inflammatory bowel disease, presence of an active acute or chronic infection, human immunodeficiency virus, or viral hepatitis, the use of immunosuppressives such as azathioprine, prednisone, or cyclosporine in the prior 4 weeks, and planned surgical resection within 2 weeks. The protocol was approved by the Duke University Medical Center Institutional Review Board, and all patients provided signed informed consent before enrollment.
Administration of Flt3L
Surgical Procedure
Collection of Specimens for Analysis On the day after the last dose of Flt3L, a 2-hour leukapheresis was performed to obtain PBMCs for analysis, and the PBMCs were cryopreserved. In addition, repeat skin testing with the recall antigens was performed. The longest diameters of induration and erythema at the skin test sites were measured.
Immunofluorescence Staining and Fluorescence-Activated Cell Sorting Analysis An alternative strategy to enumerate DCs was undertaken in some samples. Specifically, lineage markers (CD3, CD14, CD19, and CD56) were simultaneously added in one color, and the cells were counterstained with antibodies to HLA-DR, CD11c, and CD123 (IL-3R-alpha). Labeled cells were washed with phosphate-buffered saline containing 2% fetal calf serum and fixed with 1% paraformaldehyde. More than 10,000 events were collected on a FACSCaliber flow cytometer (Becton Dickinson). Data were analyzed using CellQuest software (Becton Dickinson). In all experiments, isotypically stained cells were used to set cursors so that less than 1% of the cells were considered positive.
Immunopathologic Analysis of Tumor Specimens
Statistical Analysis
Patient Characteristics Of the 12 patients enrolled onto the study, there were equal numbers of men and women and the median age was 63 years. Ten patients had received prior chemotherapy ranging from 1 to 26 months (median, 3.5 months) before initiation of Flt3L. Seven patients had liver metastases and five had lung metastases. Ten of the 12 were deemed by their surgeon to be candidates for possible resection of metastatic disease, although two of these 10 had lesions for which the likelihood of successful resection was believed to be low. Neither of these two patients ultimately proceeded to surgery. The remaining two of 12 were thought more likely to be candidates for another surgical procedure (placement of an intrahepatic arterial infusion pump). All eight patients initially thought to have a high likelihood of resectability had complete resections of their metastatic tumors. One patient underwent resection of a pulmonary lesion and then received Flt3L before undergoing resection of a contralateral metastasis. Another patient received Flt3L before each of two procedures to resect separate pulmonary metastases. As described above, resections were performed at a median of 2 days after the last Flt3L injection.
Administration and Tolerability of Flt3L
Hematologic Response
Phenotypic Analysis of Flt3L-Mobilized PBMCs Phenotypic analysis of PBMCs before and after Flt3L administration demonstrated a significant increase in circulating DCs. Figure 2 depicts the results for a representative patient. The percentage of myeloid DCs, defined as the CD11c+CD14- PBMCs, increased from 2.4% ± 1.8% to 8.8% ± 4.7% (P = .004), and the percentage of CD11c+CD14+ monocytes/DC precursors increased from 19% ± 10% to 49% ± 11% (P = .004). Given these percentages, the absolute number of DCs in 1 mL of blood increased nine-fold (range, four- to 25-fold) from 5.3 x 104 DCs/mL before Flt3L to 4.7 x 105 DCs/mL after Flt3L.
Further phenotypic analyses of the CD11c+CD14- DCs (gate R2) and CD11c+CD14+ monocytoid cells (gate R3) for markers associated with DC maturation demonstrated that both lack CD1a, CD80, and CD83 and have similar dim expression of CD40 and bright expression of HLA-DR. The lack of CD80 and CD83 expression suggests that these cells are immature. CD86 and CD11b expression, as measured by mean fluorescence intensity (MFI), was dimmer on the CD11c+CD14- cells than on the CD11c+CD14+ cells (MFI for CD86, 41 ± 25 [mean ± SD] v 237 ± 114 [P = .001]; MFI for CD11b, 369 ± 78 v 2,524 ± 630 [P = .00006], respectively), whereas HLA-DR expression was brighter on the CD11c+/CD14- cells (420 ± 189 v 171 ± 96 [P = .0003]). Although a small percentage of the cells had adherent T cells (as demonstrated by coexpression of CD3), the number of cells expressing CD4 was greater than accounted for by adherent T cells, demonstrating that CD4 was similarly expressed by both CD11c+CD14- and CD11c+CD14+ cells. An alternative analysis for DCs used detection of CD11c+ CD14-IL-3Rdim myeloid DC and the CD11c-CD14-IL-3Rbright plasmacytoid DC populations. In independent analyses of PBMCs for these markers (Fig 3) in three patients, cells were stained with a lineage marker cocktail (CD3, CD14, CD19, and CD56), as well as with CD11c, HLA-DR, and CD123. By gating on the lineage-negative, HLA-DR+ cells, we confirmed the presence of these two populations of DCs, CD11c+IL-3R- or dim myeloid DCs and CD11c-IL-3Rbright plasmacytoid DCs. Before Flt3L, these two populations were found in approximately equal proportions (43% v 38% of the lineage-negative, HLA-DR+ cells), but after Flt3L mobilization, the CD11c+IL-3R- DCs outnumbered the CD11c-IL-3R+ DCs (75% v 19%). Although the absolute number of both populations increased, this suggests that the CD11c+IL-3R- or dim DCs are preferentially mobilized by Flt3L. Comparison of the number of lineage-negative CD11c+IL-3R- or dim DCs with the number of large, CD11c+CD14- cells suggests that the two gating strategies yield counts of similar magnitude. Using four-color fluorescence, we found that the HLA-DR+CD11c+CD123- cells identified the same population as did the CD11c+CD14- cells (data not shown).
Recall Antigen Responses We found that DTH reactivity increased after Flt3L mobilization. We administered intradermal injections of standard recall antigen preparations of Candida, mumps, Trichophyton, and tetanus toxoid and the primary tumor antigen CAP-1 before and after the Flt3L. In Fig 4, the diameter of erythema of each injection site is plotted for each patient. The DTH responses showed marginally significant increases in reactivity for three of the antigens after Flt3L administration (Candida, P = .06; mumps, P = .03; tetanus, P = .08), but they were not significant for Trichophyton (P = .31) or CAP-1 (P = .13). One patient developed widespread erythema of the volar aspect of the forearm and induration at the post-Flt3L mumps injection site. Only one patient had no DTH reactivity before or after Flt3L. Increases in DTH reactivity did not seem to be related to the amount of increase in peripheral-blood DCs or infiltration of DCs into tumors.
DC, T-Cell, and NK-Cell Infiltration Into Tumor Sites We found a trend toward increased DC infiltration in the peritumoral tissue after Flt3L mobilization. We had hypothesized that administration of Flt3L would lead to DC, T-cell, and NK-cell infiltration into tumors. Therefore, specimens of tumor and uninvolved tissue from the surgical resection of eight patients (four with liver metastases and four with lung metastases) were stained for the DC markers S-100, fascin, and CD1a, the T-cell markers CD4 and CD8, and the NK-cell marker CD56. Six specimens (four liver and two lung metastases) from patients who had not received Flt3L were used as controls. The characteristics of these control patients were similar to those of the study patients (median age, 56 and 63 years, respectively; equally good performance status; median time since prior chemotherapy, 6.5 and 3.5 months [P = .85], respectively; median tumor volume, 18.8 cm3 and 8.7 cm3 [P = .65], respectively). Because they had not received any cytokines before surgery, control patients had a median WBC count of 5.1 x 103/mm3 and the percentage of monocytoid cells in the peripheral blood was 6%, similar to that of the study patients before they received Flt3L. The median number of fascin-positive (Fig 5A) and S-100-positive cells (Fig 5B) detected in the periphery of the tumor and peritumoral tissue (the zone immediately surrounding the tumor, within one to two high-power fields) tended to be greater in patients who had received Flt3L, although there was considerable overlap. Examples of the pathologic specimens are presented in Fig 6. CD1a+ cells were rarely seen in any patient (data not shown). In general, there were few DCs present in the center of the tumor masses, especially in the pulmonary metastases. Instead, they were concentrated in the periphery and the parenchymal tissue surrounding the tumor. The number of DCs was greater in the tumor periphery than in uninvolved tissue away from the tumor, regardless of whether the patient received Flt3L. Although uninvolved liver tissue was noted to contain DCs, pulmonary tissue did not. One patient with multiple pulmonary metastases who underwent resection then received Flt3L followed by another resection had an increase (nearly two-fold) in the number of fascin-positive DCs in the second specimen. Another patient who received Flt3L followed by a pulmonary metastasis resection and then another cycle of Flt3L followed by a second resection had a three-fold increase in the number of fascin-positive cells in the periphery of the tumor from the second specimen. In all patients, CD4 and CD8 cells were too numerous to count, regardless of Flt3L administration. CD4+ cells seemed to outnumber CD8 cells by five to 15 times. Few CD56+ cells (five to 15 total in five high-power fields) were found in any patient, and there were no obvious differences between those who received Flt3L and those who did not.
Clinical Response In the seven patients who had imaging of the tumor after Flt3L, no objective responses were documented. One patient with an elevation of the CEA tumor marker and one with a borderline elevated CEA level had small declines in their CEA levels after the Flt3L. The remainder had stable or increased CEA levels. The median CEA level was 7.7 ng/mL before Flt3L and 8.7 ng/mL after Flt3L. With a median follow-up of 300 days, six of the eight patients who underwent resections had recurrences at a median period of 256 days.
We demonstrated that the preoperative administration of Flt3L to patients with metastatic colon cancer is safe and does not interfere with surgical resection and postoperative care. Flt3L mobilization resulted in an increase in total WBCs and an increase in DC number in the peripheral blood with a trend toward an increase in DCs infiltrating peritumoral tissue. In addition, we observed that immune function, as determined by DTH responses against recall antigens, was increased after Flt3L. Other studies, reported in abstract form, have demonstrated an increase in DCs in peripheral blood after Flt3L. In the initial dose escalation study,15,16 doses of 10 to 100 µg/kg/d were administered to healthy volunteers for 14 days, with skin reactions and lymphadenopathy the only reported adverse events. A five-fold increase in the number of monocytes and a 30-fold increase in the number of CD11c+ DCs (averaging 8 x 105/mL of blood) were observed at day 9, similar to our results at day 14. Pulendran et al17 administered Flt3L 10 µg/kg/d for 10 days to healthy volunteers and observed a 48-fold increase in CD11c+IL-3R- DCs and a 13-fold increase in the CD11c-IL-3R+ DCs (previously called plasmacytoid T cell–derived DCs), which was greater than what was observed in our patients. In a separate abstract,18 the same group reported two of the volunteers experienced the grade 1 toxicity of low-grade fever, node tenderness, and erythema at the injection site (as we observed in one patient who was given three consecutive injections into the same site). Gasparetto et al19 randomized patients with high-risk breast cancer to receive Flt3L (50 µg/kg/d for 3 days) plus GM-CSF (10 µg/kg/d for 9 to 10 days), Flt3L (50 µg/kg/d for 3 to 8 days) plus G-CSF (10 µg/kg/d for 8 to 11 days), or G-CSF (10 µg/kg/d for 5 to 9 days). The total yield of DCs, as defined by lineage (CD3, CD14, CD19, and CD56)-negative, CD11c+, HLA-DR+ cells, was greater in the Flt3L groups than in the G-CSF group, although the number of DCs per unit volume of blood was not reported. Phenotypic analysis of the mobilized DCs demonstrated expression of CD11b, CD18, CD33, CD38, CD45, CD54, low levels of CD86, and no CD83, similar to our observations. Comparison of the results of DC mobilization in healthy donors with those in our cancer patients suggests a two-fold greater ability to mobilize DCs in healthy individuals. A possible explanation for this difference in the study patients compared with healthy volunteers is their older age and, thus, less robust mobilization. Bulky tumor burden or the cytokine milieu in cancer patients may also reduce the differentiation of precursors to DCs,20,21 although this is more likely a local rather than systemic effect. Alternatively, the DCs circulating in the peripheral blood may not represent a true measure of the number of DCs induced by the Flt3L therapy, especially if large numbers of DCs are found to infiltrate tumors in cancer patients. Differences in flow cytometry procedures and reagents may also account for part of the difference in results. We evaluated DCs in two ways in some patients, by gating on large CD11c+CD14- cells and also by gating on lineage-negative HLA-DR+ CD11c+ CD123 dim or -negative cells. These strategies gave results of a similar magnitude. Finally, we cryopreserved each specimen so that we could analyze the mobilized and unmobilized specimens concurrently. It is possible that cryopreservation decreases the percentage of viable DCs, but it is expected that it would have a similar impact on the percentage of cells from all time points. Our study is the first to administer Flt3L in the preoperative setting, allowing us to evaluate DC infiltration into tumors after Flt3L administration. There was a trend toward greater DC infiltration in those individuals who received Flt3L. This is consistent with murine studies that have documented the infiltration of tumors4,5 with DCs after Flt3L administration. Because there is a correlation between DC infiltration and survival in some human malignancies,22 this finding could have therapeutic implications. Most patients underwent surgical resection within a few days of completing the Flt3L therapy, during a time when the peripheral-blood DC count was still elevated. Thus, we believe the cell counts obtained from the surgical specimens are reflective of the DC infiltration during Flt3L administration. Nonetheless, the kinetics of DC infiltration into tumors is not well described, and it is not known whether there is a more appropriate time to sample the tumor for DC infiltration. Since all but two patients proceeded to surgery within 2 days, we were unable to identify any obvious correlation between time to surgery and DC infiltration. We also found no obvious correlation between number of cycles of Flt3L and amount of DC infiltration into tumors, although we were intrigued by the observation that one patient who had tumor resected after each of two cycles of Flt3L had an increase in DCs in the second resection specimen. We observed that some tumors lacked significant DC infiltration into the center of the tumor compared with the infiltration in the periphery of the tumor or immediate peritumoral tissue (within one to two high-power fields from the tumor edge). Bell et al23 evaluated DC infiltration into breast adenocarcinoma tissue derived from patients not exposed to cytokines. They detected immature DCs expressing CD1a within the tumor bed but mature DCs in two thirds of the samples, confined to the peritumoral areas. Our DC staining strategy did not allow a determination of whether the infiltrating DCs were mature or immature, but we did not detect more than a minority of CD1a+ DCs in any of our specimens, whether derived from patients who received Flt3L or controls. This is consistent with the lack of CD1a expression by circulating peripheral-blood DCs after Flt3L administration. Furthermore, the circulating DCs were phenotypically immature, as determined by lack of expression of CD80 and CD83. Because normal tissue was less infiltrated with DCs than the tumors or peritumoral areas, we believe that DCs have mechanisms for homing toward tumors that remain active during Flt3L mobilization. We observed that some measures of nonspecific, T cell–mediated immune responses tended to be increased after Flt3L administration. Using a panel of recall antigens, we observed a marginally significant increase in the magnitude of the DTH response to the mumps antigen and trends toward increases of the other antigens (Candida, tetanus, and Trichophyton). Interestingly, some patients had increases in DTH reactivity against a relevant antigen, the CEA (peptide CAP-1), which was overexpressed by their tumors. The PBMCs from one of these patients who was HLA-A2+ was analyzed using peptide major histocompatibility complex (MHC) tetramers specific for T-cell receptors capable of binding CAP-1. A small increase in CAP-1 peptide-MHC-tetramer–positive T cells was observed in this patient after Flt3L administration (data not shown). Thus, while the immune responses are not specific to any single antigen, it may be possible to use Flt3L to bolster immune immunity as part of vaccination strategies if it is given along with a specific immunogen. Because of the large number of T cells infiltrating the tumor tissue in all patients, we could not determine whether T-cell numbers or subsets increased after Flt3L. In peripheral blood, there was not a significant change in total lymphocyte numbers after Flt3L; however, analysis of one sample did reveal an increase in NK-T cells (data not shown). NK cells represented a minority of the tumor-infiltrating cells. In summary, preoperative Flt3L was well tolerated by cancer patients and led to increased numbers of DCs in the peripheral blood. In addition, there were trends toward increases in cell-mediated immune responses in vivo. This suggests that studies of immunization strategies combining Flt3L mobilization and immunization are warranted. Further studies to elucidate the subset of DCs within the Flt3L-mobilized PBMCs that is most applicable to active immunotherapy strategies are also warranted. Future studies combining Flt3L with other cytokines, such as IL-12, which increases antitumor activity,7 or CD40 ligand, which results in greater numbers of DCs in murine models24 and which may serve to mature the mobilized, immature DCs, are also eagerly awaited. Finally, we have initiated studies of Flt3L-mobilized DCs, matured and loaded ex vivo with tumor antigens as an immunotherapy strategy.
Supported by Immunex, Corporation, Seattle, WA. M.A.M. is a recipient of an American Society of Clinical Oncology Career Development Award and supported by National Institutes of Health grant no. M01RR00030. We thank Doris Coleman and Miriam Chitty, RN, for their care of the patients, Mayumi Kataoka and David Snyder for performing the fluorescence-activated cell sorter analysis, and Kimberly Basden for technical assistance.
Portions of the data in this article were presented in abstract form: Morse M, Nair S, Fernandez-Casal M, et al: Flt3-ligand (FLT3L) mobilization of dendritic cells (DC) in patients with metastatic colon cancer. Blood 94:48a, 1999 (suppl 1, abstr 199).
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Chen K, Braun S, Lyman S, et al: Antitumor activity and immunotherapeutic properties of Flt3-ligand in a murine breast cancer model. Cancer Res 57: 3511-3516, 1997
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Esche C, Subbotin VM, Maliszewski C, et al: Flt3 ligand administration inhibits tumor growth in murine melanoma and lymphoma. Cancer Res 58: 380-383, 1998 5. Lynch DH, Andreasen A, Maraskovsky E, et al: Flt3 ligand induces tumor regression and antitumor immune responses in vivo. Nat Med 3: 625-631, 1997[Medline] 6. Lynch DH: Induction of dendritic cells (DC) by Flt3 ligand (FL) promotes the generation of tumor-specific immune responses in vivo. Crit Rev Immunol 18: 99-107, 1998[Medline]
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Peron JM, Esche C, Subbotin VM, et al: Flt3-ligand administration inhibits liver metastases: Role of NK cells. J Immunol 161: 6164-6170, 1998
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Maraskovsky E, Brasel K, Teepe M, et al: Dramatic increase in the numbers of functionally mature dendritic cells in flt3 ligand-treated mice: Multiple dendritic cell subpopulations identified. J Exp Med 184: 1953-1962, 1996 9. Shurin MR, Pandharipande PP, Zorina TD, et al: FLT3 ligand induces the generation of functionally active dendritic cells in mice. Cell Immunol 179: 174-184, 1997[Medline] 10. McKenna HJ: Generating a T cell tumor-specific immune response in vivo: Can flt3-ligand-generated dendritic cells tip the balance? Cancer Immunol Immunother 48: 281-286, 1999[Medline]
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Shaw SG, Maung AA, Steptoe RJ, et al: Expansion of functional NK cells in multiple tissue compartments of mice treated with flt3-ligand: Implications for anti-cancer and anti-viral therapy. J Immunol 161: 2817-2824, 1998 12. Braun SE, Chen K, Blazar BR, et al: Flt3 ligand antitumor activity in a murine breast cancer model: A comparison with granulocyte-macrophage colony-stimulating factor and a potential mechanism of action. Hum Gene Ther 10: 2141-2151, 1999[Medline]
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Pulendran B, Smith JL, Jenkins M, et al: Prevention of peripheral tolerance by a dendritic cell growth factor: Flt3 ligand as an adjuvant. J Exp Med 188: 2075-2082, 1998
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Pulendran B, Smith JL, Caspary G, et al: Distinct dendritic cell subsets differentially regulate the class of immune response in vivo. Proc Natl Acad Sci U S A 96: 1036-1041, 1999 15. Lebsack ME, McKenna JA, Hoek R, et al: Safety of Flt3 ligand in healthy volunteers. Blood 90: 170a, 1997 (suppl 1, abstr 751) 16. Marakovsky E, Roux E, Teepe M, et al: Flt3 ligand increases peripheral blood dendritic cells in healthy volunteers. Blood 90: 581a, 1997 (suppl 1, abstr) 17. Pulendran B, Burkeholder S, Kraus E, et al: Differential mobilization of distinct DC subsets in vivo by Flt3-ligand and G-CSF. Blood 94: 213a, 1999 (suppl 1, abstr) 18. Fay J, Palucka K, Pulendran B, et al: In vivo mobilization of dendritic cell precursors in normal volunteers after FLT3-L administration. Blood 94: 379a, 1999 (suppl 1, abstr) 19. Gasparetto C, Rooney B, Gasparetto M, et al: Mobilization of dendritic cells from patients with breast cancer using Flt3-ligand and G-CSF or GM-CSF. Blood 94: 636a, 1999 (suppl 1, abstr) 20. Buelens C, Verhasselt V, De Groote D, et al: Interleukin-10 prevents the generation of dendritic cells from human peripheral blood mononuclear cells cultured with interleukin-4 and granulocyte/macrophage-colony-stimulating factor. Eur J Immunol 27: 756-762, 1997[Medline]
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Ohm JE, Shurin MR, Esche C, et al: Effect of vascular endothelial growth factor and FLT3 ligand on dendritic cell generation in vivo. J Immunol 163: 3260-3268, 1999 22. Zeid NA, Muller HK: S100 positive dendritic cells in human lung tumors associated with cell differentiation and enhanced survival. Pathology 25: 338-343, 1993[Medline]
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Borges L, Miller RE, Jones J, et al: Synergistic action of fms-like tyrosine kinase 3 ligand and CD40 ligand in the induction of dendritic cells and generation of antitumor immunity in vivo. J Immunol 163: 1289-1297, 1999 Submitted February 9, 2000; accepted June 28, 2000.
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Copyright © 2000 by the American Society of Clinical Oncology, Online ISSN: 1527-7755. Print ISSN: 0732-183X
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ABSTRACT
INTRODUCTION
), lymphocytes (
), and monocytoid cells (
) before, during (days 1 through 15), and after Flt3L administration in cycle 1. *P < .01.
