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Vol. 4, 2709-2716, November 1998 Clinical Cancer Research 2709
Dendritic Cell-based Vaccines in the Setting of Peripheral Blood
neic PBSCT) and from intermediate grade non-Hodgkin’s
lymphoma or multiple myeloma patients receiving cyclo-
phosphamide plus G-CSF (for autologous PBSCT). High
enrichment of CD34�’ HSCs was obtained using an immu-
nomagnetic bead cell separation device. After separation,
the negative fraction of mobilized PBMCs from normal
donors and cancer patients contained undetectable levels of
CD34� HSCs by flow cytometry. This fraction of cells was
then subjected to plastic adherence, and the adherent cells
were cultured for 7 days in GM-CSF (100 ng/ml) and inter-
leukin 4 (50 nglml) followed by an additional 7 days in
GM-CSF, interleukin 4, and tumor necrosis factor a (10
ng/ml) to generate DCs. Harvested DCs represented yields of
4.1 ± 1.4 and 5.8 ± 5.4% of the initial cells plated from the
CD34� cell-depleted mobilized PBMCs of normal donors
and cancer patients, respectively, and displayed a high level
expression of CD8O, CD86, HLA-DR, and CD1 ic but not
CD14. This phenotypic profile was similar to that of DCsderived from non-CD34� cell-depleted mobilized PBMCs.DCs generated from CD34� cell-depleted mobilized PBMCs
elicited potent antitetanus as well as primary allogeneic
T-cell proliferative responses in vitro, which were equivalent
to DCs derived from non-CD34� cell-depleted mobilized
PBMCs. Collectively, these results demonstrate the feasibil-
ity of obtaining both DCs and CD34� HSCs from the same
leukapheresis collection from G-CSF-primed normal donors
and cancer patients in sufficient numbers for the purpose of
combined PBSCT and immunization strategies.
INTRODUCTIONDCs3 are highly potent APCs of BM origin (I ). which have
been shown to stimulate both primary and secondary T- and
B-cell responses (2, 3). We (4-7) and others (8-10) have shown
that tumor-pulsed DC-rich preparations can stimulate specific
T-cell reactivity in vitro and serve as potent antitumor vaccines
in VitO. Moreover, methods are now available to generate siz-
able numbers of highly enriched DCs in both rodents and
humans by culturing progenitor cells in the presence of GM-
CSF, TNF-a, and/or IL-4 ( 1 1-1 3). The establishment of DC
cultures from the peripheral blood of adult patients has raised
the very important possibility of now using these cells as an
immunotherapeutic agent for the treatment of a variety of hu-
man tumors (9, 14, 15).
Another form of adoptive immunotherapy involves the use
of BMT or PBSCT. This approach is currently being used for
the treatment of patients with cancer, including those with
lymphorna, leukemia, and metastatic breast cancer. Well-estab-
lished models exist in mice of successful transplantation and
hernatolymphoid reconstitution with whole, unfractionated BM
or with highly selected HSCs (16, 17). Investigators have also
Received 4/13/98; revised 8/20/98: accepted 8/26/98.The costs of publication of this article were defrayed in part by thepayment of page charges. This article must therefore be hereby markedadvertisement in accordance with I 8 U.S.C. Section 1734 solely to
indicate this fact.
‘ Supported by NIH Grants 1 P01 CA59327 and M01-RR00042 (to the
University of Michigan General Clinical Research Center), Department
of DefenseIUSAMRMC Grant DAMD17-96-l-6l03, and United StatesArmy Research Office Grant DAAG55-97-l-0239.2 To whom requests for reprints should be addressed, at the Departmentof Surgery, University of Michigan Medical Center, 1520c MSRB-l,1 150 West Medical Center Drive, Ann Arbor. MI 48109-0666. Phone:(734) 647-2779; Fax: (734) 763-4135.
3 The abbreviations used are: DC, dendritic cell; HSC, hematopoieticstem/progenitor cell; PBSCT, peripheral blood stem cell transplantation:
Fig. I FACS phenotypic profile of DCs generated from the CD34�HSC-depleted negative fraction of separated mobilized PBMCs. TheseDCs typically expressed the costimulatory molecules CD8O and CD86as well as HLA-DR but did not express CDI4. In addition, CDla,
CD1 lc, CD13, CD16, CD32, and CD33 markers were expressed atvarying levels. DCs were analyzed after a 14-day culture of the CD34�
HSC-depleted mobilized PBMCs in human recombinant GM-CSF, IL-4,
and TNF-a as described in “Materials and Methods.” Staining was
detected by a panel of antihuman FITC- or PE-labeled antibodies.
Positively stained cells are displayed by the open histograms compared
to isotype-matched control mAbs (shaded histograms). The X axis is a
logarithmic scale of fluorescence intensity, and the Y axis representscounts.
Different numbers of DCs were cultured with a fixed number of
allogeneic T cells from different donors to achieve DC stimulator:
T-cell responder ratios of 1 : 10 and I :20. These ratios were found to
be optimal for inducing T-cell alloproliferative responses in our
previous studies (data not shown). As shown by the separate
representative experiments, DCs displayed potent capacity to stim-
ulate primary allogeneic MLRs in vitro. We also evaluated the
capacity of DCs generated from the CD34� cell-depleted fraction
of the mobilized PBMCs to stimulate an autologous T-cell response
to the soluble, defined antigen iT. Fig. 3 shows a representative
experiment of potent and specific anti-TI’ T-cell proliferative re-
activity to antigen-pulsed autologous DCs from a multiple my-
eloma patient.
DCs Generated from CD34� Cell-depleted Mobilized
PBMCs Do Not Differ from DCs Generated from Non-
CD34� Cell-depleted Mobilized PBMCs by Phenotype orAllostimulatory Capacity. Next we directly compared DCs
generated from unmanipulated mobilized PBMCs versus those
Table 1 Generation of DCs from CD34� cell-depleted mobilized
PBMCs
PBMC source Donor DC yield (%)“
Normal JB 18.72
JW 5.00LD 4.62OJ 6.05DS 5.07CB 2.22
Mean ± SD 5.78 ± 5.44Lymphomab PE’� 3.08
Ki 6.92WT’ 4.70WTC 3.42
WT� 3.70
Myelorna JRC 2.88
Mean ± SD 4.06 ± 1.39
a The DC yield was determined morphologically by typical veiled
appearance; by FACS analysis, these cells also coexpressed high levels
of the costimulatory molecules CD8O and CD86 as well as HLA-DR and
CD83, but they did not express CD14.a Intermediate-grade NHL patients.
C Mobilized PBMCs from these patients were selected for CD34�
cells using the clinical scale Isolex-300 i; all others were selected with
the research scale Isolex-50.
generated from the CD34� cell-depleted negative fraction of
mobilized PBMCs phenotypically and functionally. Fig. 4
shows the FACS analysis conducted on DCs from the mobilized
PBMCs of a NHL patient. Comparable levels of cell surface
expression were noted for CD1a, CD86, CD1 ic, CD8O, and
HLA-DR (and little, if any, detectable CD14 expression) be-
tween DC cultures generated from CD34� cell-depleted
PBMCs versus those from nondepleted mobilized PBMCs. High
levels of CD83 expression were also present on both sources of
DCs, indicating a mature stage (data not shown; Refs. 25 and
26). The capacity of these two sources of DCs to stimulate a
primary allogeneic MLR was also compared. As shown in Fig.
5, comparable levels of DC allostimulatory activity were ob-
tamed, regardless of whether the DCs were generated from
CD34� cell-depleted versus nondepleted mobilized PBMCs. Of
note, the level of allostimulation of responding T cells to DCs
(at a 10: 1 ratio) was nearly equivalent to the maximum prolif-
eration of T cells obtainable by PHA stimulation. When com-
parisons of DC yields were made, the CD34� cell-depleted
fraction tended to generate slightly greater numbers of DCs
compared to similar aliquots of unmanipulated mobilized
PBMCs from both normal donors and cancer patients (data not
shown).
DISCUSSION
We and others have shown that DCs pulsed with tumor-
associated antigen(s) in the form of whole cell lysates (4-7),
peptides (8, 14), proteins (28), RNA (29), or DNA (30) could
initiate primary MHC class I- or 11-restricted T-cell responses
that resulted in antitumor effects both in vitro and in vivo. Based
on these studies, attention has focused on the use of DCs to
enhance the host immune response to tumor-associated anti-
gen(s) in clinical vaccine strategies in humans with cancer (14,
31). We have recently initiated Phase I clinical trials of autol-
Fig. 2 DCs generated from the CD34� HSC-depleted negative fractionof separated mobilized PBMCs are stimulatory in a primary 5-dayallogeneic MLR. Mobilized PBMCs from a normal donor (top panel)
and a NHL patient (bottom panel) were used to generate DCs: twoseparate mobilized PBMC donors served as the source of responder i
cells in the assay, as described in “Materials and Methods.” Stimulator
(DC):responder (i cell) ratios were I : 10 and 1:20. DCs were generatedin GM-CSF-, IL-4-, and TNF-a-supplemented cultures and tested at 14
days. The SD of replicate wells is shown by the error bars.
ogous tumor lysate-pulsed DCs as a vaccine in adult and pedi-
atric patients with advanced solid tumors. Efforts to expand this
strategy to the setting of PBSCT are now underway. Thus, the
identification of approaches that would generate large numbers
of DCs with potent antigen-presenting capacity, particularly in
the setting of PBSCT, would be an important step in the further
refinement of such a vaccine strategy.
In the current study, we focused attention on strategies
to obtain functional DCs from the same mobilized PBMCs
leukapheresis collection used to obtain highly selected
CD34� HSCs for transplantation. We have found that both
sources of CD34� cell-depleted and nondepleted mobilized
PBMCs could generate DCs of similar phenotype and func-
tion when cultured in the recombinant human cytokines GM-
CSF, IL-4, and TNF-a. Importantly, functional DCs could be
readily generated from the cryopreserved PBMC sources
upon thawing, which allows considerable flexibility in the
timing and repetitiveness of DC-based vaccine immuniza-
tions after CD34� HSC transplantation. DCs from both mo-
bilized PBMC sources displayed the typical phenotype of
high coexpression of the costimulatory molecules CD8O and
+
-I-
- - - EI�III�DCalone 1:10 :20 1-Cells alone
Dendritic Cell / T.Cell Ratios
Fig. 3 DCs generated from the CD34� HSC-depleted negative fractionof separated mobilized PBMCs from a multiple myeloma patient pos-
sess a potent antigen-presenting function. The proliferative response of
purified autologous CD4� T cells to ‘FT antigen presented by autologous
DCs was measured at day 5 as described in “Materials and Methods.”Stimulator (DC):responder (T cell) ratios were 1: 10 and 1:20. DCs weregenerated in GM-CSF-. IL-4-, and TNF-a-supplemented cultures and
tested at 14 days. The SD of replicate wells is shown by the error bars.
CD86 as well as CD83 and HLA-DR, with little, if any,
expression of CD14. In addition, these DCs, unlike CDI4’
monocytes/macrophages, were capable of potent primary
stimulation of allogeneic T cells and presentation of the
soluble antigen TT to autologous T cells in vitro, which are
hallmark functions of this cell type. Overall, our study con-
firms that of Tarte et a!. (32) and also extends their results by
now including intermediate-grade NHL patients in the anal-
ysis.
Prior studies had identified proliferating progenitors within
the CD34� cell fraction of human BM, cord blood, and adult
PBMCs that could be driven with cytokines, particularly GM-
CSF and IL-4 with or without TNF-a, to develop into potent
DCs over a 1-2-week period in culture (1, 3, 12, 13, 31). In
addition, DCs have been derived from precursors in unfraction-
ated PBMCs as well as from CDl4� blood monocytes. How-
ever, the vast majority of functional DCs generated from non-
mobilized PBMCs have been shown to be derived from both
CDl4� and CD2� precursors ratherthan from CD34� HSCs (1,
12, 26, 33). Thus, the demonstration that functional DCs can be
generated from the CD34� cell-depleted mobilized PBMCs
adds confidence that these DCs are those that should be capable
of potent APC activity in vito. These DCs could now be used in
vaccine strategies in the setting of CD34� HSC transplants in
cancer patients.
In the transplant setting, DCs could be pulsed with
relevant idiotype proteins or whole tumor lysates that serve
as tumor-associated antigens in B-cell lymphoma and multi-
pie myeloma as well as potentially in leukemia patients who
have relapsed after allogeneic transplant. In addition, several
known tumor-associated peptides recognized by cytolytic T
cells have been molecularly cloned and shown to be shared
(or common) for certain HLA haplotypes in breast/ovarian
cancer (namely HER2/neu and carcinoembryonic antigen;
Fig. 4 Comparison of FACS phenotypic profiles of DCs generated
from CD34� HSC-depleted versus CD34� HSC nondepleted (unma-nipulated) mobilized PBMCs from a NHL patient. DCs from both
sources expressed comparable levels of the costimulatory molecules
CD8O and CD86 as well as HLA-DR but did not express CD14. In
addition, these DCs also displayed comparable levels of a series of
CDI6. CD32, CD33, CDIa, CDI3, and CD1 Ic markers, with theexception of CD14. DCs were analyzed after a 14-day culture of therespective mobilized PBMCs in human recombinant GM-CSF, IL-4,
and TNF-a as described in “Materials and Methods.” Staining was
detected by a panel of antihuman FITC- or PE-labeled antibodies.
Positively stained cells are displayed by the open histograms compared
to the isotype-matched control mAbs (shaded histograms).
Refs. 34 and 35), which would offer the possibility of ex-
panding this DC vaccine approach to solid tumors (e.g.,
breast and ovarian tumors) as well. In the allogeneic (MHC-
matched, unrelated) transplant setting, the source of DCs
would be autologous to the CD34� HSC transplant and the
resulting progeny hematolymphoid lineages upon reconstitu-
tion. The immunization of patients with peptide/protein-
pulsed DCs in the allogeneic transplant setting offers the
intriguing possibility of augmenting graft versus tumor ef-
fects without augmenting the graft versus host reaction in the
transplant recipient. It is also conceivable, however, that the
high potency of DCs in antigen-presenting function could
uncover or enhance the reactivity to minor histocompatibility
antigens, which could, in turn, increase graft-versus-host
disease.
Aside from the fact that immunomagnetic bead separa-
tion is a rapid and effective approach for isolating engrafting
doses of CD34� HSCs from mobilized PBMCs, as shown in
1:160 1:320 � 1:640alone alone + P1-IA
Dendritic Cell / T-Cell Ratios
Fig. 5 DCs generated from both CD34C HSC-depleted and CD34HSC nondepleted (unmanipulated) mobilized PBMCs from a NHLpatient are comparable in their stimulatory activity in a primary, 5-day
allogeneic MLR at varying stimulator (DC):responder (T cell) ratios.
For comparison, the maximum T-cell proliferation induced by the lectin
PHA was used as the positive control, as described in “Materials and
Methods.” The SD of replicate wells is shown by the error bars.
the current study as well as in those of others (24, 36), we
demonstrated that ample numbers of DCs can also be gener-
ated from the same leukapheresis collection by using the
CD34� cell-depleted negative fraction of mobilized PBMCs.
Importantly, culture of this negative fraction in recombinant
cytokines GM-CSF, IL-4, and TNF-a resulted in overall DC
yields of 4-5% at 14 days. In terms of theoretical clinical
scale-up, this amount would represent 4-5 X 108 DCs for
every I X lO’#{176} mobilized PBMCs plated in culture after
CD34� HSC removal. This yield should be considered sub-
stantial, given the fact that as few as a median of 5 X 106
tumor antigen-charged DCs freshly obtained from whole,
unfractionated PBMCs have been shown in an early clinical
vaccine trial in follicular B-cell lymphoma patients (not
undergoing PBSCT or BMT) to augment cellular antitumor
reactivity in 4 of 4 immunized patients and cause complete or
partial tumor regressions in 3 of 4 of those patients (37). In
a recent reported clinical study in advanced melanoma pa-
tients (in the nontransplant setting) immunization with 1 X
106 autologous DCs/injection has resulted in some partial and
complete responses as well as in the induction of tumor-
specific cytotoxic T cells (38).
Collectively, our data demonstrate the feasibility of gener-
ating potent DCs from the same leukapheresis as isolated
CD34� HSCs used for transplant purposes. The advantages of
now attempting DC-based immunizations in the setting of
PBSCT include lower tumor burden, reduction (or elimination)
of tumor-induced immunosuppression, and the possibility of
biasing or educating the developing immune T-cell repertoire to
selectively target and potentially eliminate residual malignant
disease in the transplanted patient.
ACKNOWLEDGMENTSWe thank Dr. Douglas Fraker (University of Pennsylvania, Phila-
delphia, PA) for the provision of recombinant TNF-a and Dr. Satwant
Narula, Dr. Mary Ellen Rybak, and Chris DeLuca (Schering-Plough
Research Institute, Kenilworth, NJ) for the generous supplies of recom-
binant human GM-CSF and IL-4. We also thank Dr. Larry Baker of the
University of Michigan Comprehensive Cancer Center (Ann Arbor, MI)
and Dr. Paul Watkins, Dr. Blake Roessler, and Dorene Markel of the
University of Michigan General Clinical Research Center (Ann Arbor,
MI) for their efforts in establishing a dedicated leukapheresis and HSC
separation facility for clinical research studies. The provision of
Isolex-50 and Isolex-300 i devices by Dr. Dennis Van Epps and Baxter
Healthcare Corp. is greatly appreciated.
REFERENCES
I . Steinman, R., and Nussenzweig, M. Dendritic cells: features and
functions. Immunol. Rev., 53: 127-147, 1980.
2. Steinman, R. M., Gutchinor, B., Witmer, M. D., and Nussenzweig,M. C. Dendritic cells are principal stimulators of the primary mixedleukocyte reaction in mice. J. Exp. Med., /57: 613-627, 1983.
3. Stingl, G., and Bergstresser, P. R. Dendritic cells: a major story
unfolds. Immunol. Today, 16: 330-333, 1995.
4. Geraghty, P. J., Fields, R. C., and Mule, J. J. Vaccination with
tumor-pulsed splenic dendritic cells mediates immunity to poorly-im-
munogenic tumor. Surg. Forum, 47: 459-461, 1996.
5. Cohen, P. J., Cohen, P. A., Rosenberg, S. A., Katz, S. I., and Mule,J. J. Murine epidermal Langerhans cells and splenic dendritic cellspresent tumor-associated antigens to primed T cells. Eur. J. Immunol.,
24: 315-319, 1994.
6. Cohen, P. A., Cohen, P. J., Rosenberg, S. A., and Mule, J. J. CD4�T-cells from mice immunized to syngeneic sarcomas recognize distinct,non-shared tumor antigens. Cancer Res., 54: 1055-1058, 1994.
7. Cohen, P. A., Kim, H., Fowler, D. H., Gress, R. E., Jakobsen, M. K.,Alexander, R. B., Mule, J. J., Carter, C., and Rosenberg, S. A. Use ofinterleukin-7, interleukin-2, and interferon--y to propagate CD4� T cellsin culture with maintained antigen specificity. J. Immunother., 14:
242-252, 1993.
8. Mayordomo, J. I., Zorina, Y., Storkus, W. J., Zitvogel, L., Celluzzi,
C., Falo, L. D., Melief, C. J., Ildstad, S. T., Kast, W. M., DeLeo, A. B.,and Lotze, M. T. Bone marrow-derived dendritic cells pulsed withsynthetic tumor peptides elicit protective and therapeutic antitumor
immunity. Nat. Med., I: 1297-1302, 1995.
9. Grabbe, S., Beissert, S., Schaw, T., and Granstein, R. D. Dendriticcells as initiators of tumor immune responses: a possible strategy for
10. Flamand, V., Sornasse, T., Thielemans, K., Demanet, C., Bakkus,M., Basin, H., Thielemans, F., Leo, 0., Urbain, J., and Moser, M.Murine dendritic cells pulsed in vitro with tumor antigen induce tumor
resistance in vivo. Eur. J. Immunol., 24: 605-610, 1994.
1 1 . Sallusto, F., and Lanzavecchia, A. Efficient presentation of soluble
antigen by cultured human dendritic cells is maintained by granulocyte/
macrophage colony-stimulating factor plus interleukin 4 and down-regulated by tumor necrosis factor-a. J. Exp. Med., 179: 1109-1118.
1994.
12. Romani, N., Gruner, S., Brang, D., Kampgen, E., Lenz, A., Trock-enbacker, B., Konwalinka, G., Fritsch, P. 0., Steinman, R. M., and
Schuler, G. Proliferating dendritic cell progenitors in human blood. J.Exp. Med., 180: 83-93, 1994.
13. Grabbe, S., Bruvers, S., Lindgren, A. M., Hosoi, J., Tan, K. C., and
Granstein, R. D. Tumor antigen presentation by epidermal antigen-
presenting cells in the mouse: modulation by granulocyte-macrophage
colony-stimulating factor, tumor necrosis factor a, and ultraviolet radi-
ation. J. Leukoc. Biol., 52: 209-217, 1992.
14. Lotze, M. 1. Getting to the source: dendritic cells as therapeutic
reagents for the treatment of patients with cancer. Ann. Surg., 226: 1-5,
1997.
15. Morse, M. A., Zhou, L. J., Tedder, T. F., Lyerly, H. K., and Smith,C. Generation of dendritic cells in vitro from peripheral blood mono-
nuclear cells with granulocyte-macrophage-colony-stimulating factor,
interleukin-4, and tumor necrosis factor-a for use in cancer immuno-
therapy. Ann. Surg., 226: 6-16, 1997.
16. Morel, F., Szilvassy, S. J., Travis, M., Chen, B., and Galy, A.
Primitive hematopoietic cells in murine bone marrow express the CD34
antigen. Blood, 88: 3774-3784, 1996.
17. Uchida, N., and Weissman, I. L. Searching for hematopoietic stem
cells: evidence that Thy-l.l lo Lin-Sca-l + cells are the only stem cells
in C57BL/Ka-Thy-l.l bone marrow. J. Exp. Med., 175: 175-184, 1992.
18. Katsanis, E., Xu, Z., Anderson, P. M., Dancisak, B. B., Bausero,
M. A., Weisdorf, D. J., Blazar, B. R., and Ochoa, A. C. Short-term ex
t,ivo activation of splenocytes with anti-CD3 plus IL-2 and infusion
post-BMT into mice results in in vivo expansion of effector cells withpotent anti-lymphoma activity. Bone Marrow Transplant., 14: 563-572,
1994.
19. Slavin, S., Naparstek, E., Nagler, A., Ackerstein, A., Samuel, S.,
Kapelushnik, J., Brautbar, C., and Or, R. Allogeneic cell therapy with
donor peripheral blood cells and recombinant interleukin-2 to treatleukemia relapse after allogeneic bone marrow transplantation. Blood,
87: 2195-2204, 1996.
20. Kwak, L. W., Pennington, R., and Longo, D. L. Active immuniza-tion of murine allogeneic bone marrow transplant donors with B cell
tumor-derived idiotype: a strategy for enhancing the specific antitumor
effect of marrow grafts. Blood, 87: 3053-3060, 1996.
21. Mackall, C. L., Bare, C. V., Granger, L. A., Sharrow, S. 0.. Titus,J. A., and Gress, R. E. Thymic-independent T cell regeneration occursvia antigen-driven expansion of peripheral T cells resulting in a reper-
toire that is limited in diversity and prone to skewing. J. Immunol., 156:
4609-4616, 1996.
22. Vavassori, M., Maccario, R., Moretta, A., Comoli, F., Casorati,G., and Montagna, D. Restricted TCR repertoire and long-termpersistence of donor-derived antigen-experienced CD4� T cells inallogeneic bone marrow transplantation recipients. J. Immunol., 157:
5739-5747, 1996.
23. Ridge, J. P., Fuchs, E. J., and Matzinger, P. Neonatal tolerancerevisited: turning on newborn T cells with dendritic cells. Science
(Washington DC), 271: 1723-1726, 1996.
24. Williams, S. F., Lee, W. J., Bender, J. G., Zimmerman, T., Swinney,
P., Blake, M., Oldham, F., and Van Epps, D. Selection and expansion ofperipheral blood CD34� cells in autologous stem cell transplantation for
breast cancer. Blood, 87: 1687-1691, 1996.
25. Chen, B-G., Shi, Y., Smith, J. D., Choi, D., Geiger, J. D., andMule, J. .i. The role of tumor necrosis factor a in modulating the
quantity of peripheral blood-derived, cytokine-driven human den-
dritic cells and its role in enhancing the quality of dendritic cell
function in presenting soluble antigens to CD4� T cells in vitro.
Blood, 91: 4652-4661, 1998.
26. Zhou, L. J., and Tedder, T. F. CDl4� blood monocytes can differ-entiate into functionally mature CD83� dendritic cells. Proc. NatI. Acad.
Sci. USA, 93: 2588-2593, 1996.
27. Trizio, D., and Cudkowicz, G. Separation of T and B lymphocytesby nylon wool columns: evaluation of efficacy by functional assays in
vito. J. Immunol., 113: 1093-1097, 1974.
28. Paglia, P., Chiodoni, C., Rodolfo, M., and Colombo, M. P. Murinedendritic cells loaded in vitro with soluble protein prime cytotoxic Tlymphocytes against tumor antigen in vivo. J. Exp. Med., 183: 3 17-322,
1996.
29. Boczkowski, D., Nair, S. K., Snyder, D., and Gilboa, E. Dendritic
cells pulsed with RNA are potent antigen-presenting cells in vitro and in
vivo. J. Exp. Med., 184: 465-472, 1996.
30. Condon, C., Watkins, S. C., Celluzzi, C. M., Thompson. K., andFalo, L. D., Jr. DNA-based immunization by in vivo transfection of
dendritic cells. Nat. Med., 2: 1 122-1 128, 1996.
31. Hsu, F. J., Engleman, E. G., and Levy, R. Dendritic cells and their
application in immunotherapeutic approaches to cancer therapy. hi:
V. T. DeVita, Jr., S. Hellman, and S. A. Rosenberg (eds.), Cancer
Principles and Practice of Oncology Updates. Vol. 11, pp. 1-14. Cedar
32. Tarte, K., Lu, Z. Y., Fiol, G., Legouffe, E., Rossi, J-F., and Klein,B. Generation of virtually pure and potentially proliferating dendriticcells from non-CD34 apheresis cells from patients with multiple my-
eloma. Blood, 90: 3482-3495, 1997.
33. Takamizawa, M., Rivas, A., Fagnoni, F., Benike, C., Kosek, J.,
Hyakawa, H., and Engleman, E. Dendritic cells that process and presentnominal antigens to naive T lymphocytes are derived from CD2�
precursors. J. Immunol., 158: 2134-2142, 1997.
34. Peoples, G. E., Goedegebuure, P. 5., Smith, R., Linehan, D. C.,Yoshino, I., and Eberlein, T. J. Breast and ovarian cancer-specific
cytotoxic T lymphocytes recognize the same HER2/neu-derived pep-tide. Proc. NatI. Acad. Sci. USA, 92: 432-436, 1995.
35. Tsang, K. Y., Zaremba, S., Nieroda, C. A., Zhu, M. Z., Hamilton,J. M., and Schlom, J. Carcinoembryonic antigen epitopes from patients
immunized with recombinant vaccinia-CEA vaccine. J. Nail. Cancer
Inst., 87: 982-990, 1995.
36. Cooper, D. L. Peripheral blood stem cell transplantation. ln: V. T.DeVita, Jr., S. Hellman, and S. A. Rosenberg (eds.), Cancer Principalsand Practice of Oncology Updates, Vol. 8, pp. 1-12. Cedar Knolls, NJ:Lippincott-Raven Healthcare, 1994.
37. Hsu, F. J., Benike, C., Fagnoni, F., Liles, T. M., Czerwinski, D.,Taidi, B., Engleman, E. G., and Levy, R. Vaccination of patients with B
1998;4:2709-2716. Clin Cancer Res D Choi, M Perrin, S Hoffmann, et al. peripheral blood can serve as a source of potent dendritic cells.stem cell transplantation: CD34+ cell-depleted mobilized Dendritic cell-based vaccines in the setting of peripheral blood