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Division of Oncology, Department of Medicine, Stanford University School of Medicine, Stanford, CA 94305
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Six- to 8-wk-old female C3H/HeN mice were obtained from Harlan
Sprague-Dawley (San Diego, CA) and were housed at the Laboratory Animal
Facility at Stanford University Medical Center (Stanford, CA). The
carcinogen-induced murine B cell lymphoma 38C13 expressing a clonal
IgM/
on its surface has been previously described (16).
38C13 cells were maintained in RPMI 1640 medium supplemented with 10%
heat-inactivated FCS, 2 mM L-glutamine, 100 U/ml
penicillin, 100 µg/ml streptomycin, and 50 µM 2-ME (cRPMI-10). All
media and supplements were obtained from Life Technologies/BRL
(Frederick, MD).
Generation and Ag pulsing of bone marrow-derived DC
Bone marrow-derived DCs were prepared as previously described
(3) with minor modifications. Briefly, bone marrow was
flushed from the femurs and tibias of mice and depleted of red cells by
ammonium chloride lysis. Bone marrow cells were then depleted of T, B,
and MHC class II+ cells by treatment with rat
mAbs followed by panning on plates coated with goat anti-rat Ig Abs
(Southern Biotechnology Associates, Birmingham, AL). The mAbs used were
GK1.5 (anti-CD4), 53-6.7 (anti-CD8) (TIB 207 and TIB 105,
respectively, American Type Culture Collection, Manassas, VA), RA3-6B2
(anti-B220, PharMingen, San Diego, CA), and 10-3.6
(anti-I-Ak, PharMingen). Cells were then
plated in six-well culture plates at 106/ml in
cRPMI-10 without cytokines. After overnight culture, nonadherent cells
were harvested and replated at 2.5 x 105/ml
in cRPMI-10 supplemented with 10 mM HEPES, 1000 U/ml recombinant murine
GM-CSF (provided by Immunex, Seattle, WA), and 1000 U/ml recombinant
murine IL-4 (provided by Dr. Robert Coffman, DNAX, Palo Alto, CA).
Medium was replenished (
50%) after 4–5 days as needed. After 6
days of culture, nonadherent and loosely adherent cells were harvested
by gently pipetting and replated at 106 cells/ml
in fresh medium with cytokines. Nonadherent DCs were harvested on the
seventh day, replated at 106 cells/ml, and
cultured overnight in the presence of medium alone or Ag at 100
µg/ml. This concentration of Ag was chosen based on previous studies
in several other murine tumor models (2, 6). Except where
noted, pulsing medium was supplemented with 1000 U/ml GM-CSF. The
following day, nonadherent and loosely adherent DCs were harvested,
washed three times, and resuspended in HBSS.
Production and modification of Id proteins
38C13 Id and an isotype-matched IgM/ protein (4C5) were
derived from tumor-myeloma cell hybridomas as previously described
(16) and affinity purified using mannose binding protein
columns (Pierce, Rockford, IL) to >95% purity as determined by
SDS-PAGE. Proteins were coupled to KLH (Calbiochem, San Diego, CA)
using glutaraldehyde as previously described (16).
Chimeric Id protein, composed of 38C13 variable regions linked to human
IgG1/ constant regions (Id-HCR), and chimeric Id fusion protein
containing murine GM-CSF at its C-terminus (Id-HCR-GM) were produced as
previously described (26).
Vaccinations
Washed, Ag-pulsed DCs were injected i.v. by tail vein in a volume of 0.1 ml of HBSS. In different experiments, the number of injected DCs varied between 2–5 x 105/mouse. DC vaccines were compared with Id-HCR-GM fusion protein (50 µg) administered i.p. twice biweekly in PBS without additional adjuvants and to Id-KLH conjugate (50 µg) given s.c. twice biweekly in PBS or together with QS21 adjuvant (31) (10 µg) three times biweekly (QS21 was provided by Aquila Biopharmaceuticals, Framingham, MA).
Humoral immune response assessments
Twelve to 14 days following the last immunization, blood was
collected by tail vein. Serum anti-Id Abs were quantitated by ELISA
as previously described (16). Briefly, 96-well Maxisorb
plates (Nunc, Naperville, IL) were coated with 38C13 IgM and incubated
with serially diluted immune sera. Bound Abs were detected with
HRP-conjugated goat anti-mouse IgG (Caltag, Burlingame, CA) using
the 2,2'-azinobis-(3-ethyl)-benzthiazoline sulfonic acid substrate and
absorbance determination at 405 nm with a Vmax microplate reader
(Molecular Devices, Menlo Park, CA). A mixture of affinity-purified
anti-38C13 Id mAbs containing IgG1, IgG2a, and IgG2b isotypes in a
2:1:1 ratio was used to generate a standard curve for total anti-Id
IgG. Isotype-specific detectors were used for the quantitation of
anti-Id Abs of the IgG1 and IgG2a subclasses (Southern
Biotechnology Associates) using relevant isotype standards. Purified
4C5 (IgM/) and BCL1 (IgM/
) Igs were used to check the specificity
of the anti-Id response.
Tumor challenge
38C13 tumor cells were thawed from a common dedicated frozen stock 2 days before tumor challenge and were split on the day before use. On the day of tumor challenge, cells were washed three times in serum-free RPMI and diluted to the appropriate concentration in HBSS. Groups of mice received either 1000 cells s.c. above the base of the tail or 200 cells i.p. in a volume of 0.2 ml. Thereafter, mice were followed daily for survival. Survival analysis was performed using GraphPad Prism software (San Diego, CA), and p values were calculated using the log-rank statistical test.
In vivo depletion of T lymphocyte subsets
Groups of 10 mice were vaccinated three times biweekly with Id-KLH-pulsed DCs and challenged with tumor 2 wk later on day 0. On days -6, -5, -4, and 0 and weekly thereafter for 4 wk mice were injected i.p. with 200 µg of T cell-depleting or control mAbs (from ascites). Abs used were the CD4+ T cell-depleting mAb GK1.5 (rat IgG2b), control rat IgG2a mAb H22-15–5, CD8+ T cell-depleting mAb HB129 (mouse IgG2a), or control mouse IgG2a mAb 17F12. On day -1 with respect to tumor challenge, peripheral blood was collected from two representative mice from each group, and lymphocytes were analyzed by flow cytometry for depletion of the appropriate T cell subset. Staining Abs CT-CD4 (anti-CD4, Caltag) and 53-5.8 (anti-CD8ß.2, PharMingen) were not cross-blocked by the depleting Abs and demonstrated >98% depletion of the target T cell population.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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To identify an Ag pulsing strategy that would lead to greater
immunogenicity of tumor Ag-pulsed DC vaccines, we examined the use of
modified tumor Ag (Id) proteins for DC pulsing. To evaluate this
approach we chose bone marrow-derived DCs grown in GM-CSF and IL-4.
Such DCs have been shown to take up and process soluble protein Ags
(6, 8, 27). For the Ag, we first examined recombinant
forms of the 38C13 Id containing xenogeneic constant region and
cytokine sequences, since in previous studies these proteins had
displayed increased immunogenicity over that of native Id
(26). We compared the immunogenicity of DCs pulsed with
native Id, a chimeric Id protein containing xenogeneic (human) constant
region sequences (Id-HCR), or a chimeric Id protein also having murine
GM-CSF linked to its C terminus (Id-HCR-GM) (26). Mice
received two biweekly i.v. injections of Ag- or medium-pulsed DCs.
Following vaccination we monitored the humoral anti-Id response by
ELISA (16). The measurement of anti-Id Abs is highly
relevant in this tumor model, as previous studies have demonstrated
that the induction of humoral anti-Id immunity was sufficient to
provide protection from tumor challenge (16, 29, 32).
After two immunizations, there was no detectable humoral response in
mice vaccinated with DCs pulsed with native Id protein (Fig. 1
). Low levels of anti-38C13 Id IgG
were detected in mice given DCs pulsed with the chimeric Id, yet
substantially higher levels were elicited by DCs pulsed with the
chimeric GM-CSF fusion protein. Administration of DCs pulsed with an
irrelevant tumor Id (BCL-1) chimeric GM-CSF fusion protein failed to
elicit Abs specific for the 38C13 Id (data not shown). GM-CSF is known
to enhance the uptake and processing of Ag by DCs (6, 28).
Therefore, we next asked whether excess soluble GM-CSF present during
the pulsing period could substitute for the cytokine moiety linked
directly to Id in the Id-HCR-GM fusion protein. Soluble GM-CSF (200
ng/ml = 20-fold excess of that which supports DC growth and
differentiation in vitro) added during overnight Ag pulsing could not
enhance the humoral anti-Id response to DCs pulsed with the
chimeric Id to the level attained when using Ag directly fused to the
cytokine (0.62 ± 0.39 vs 2.7 ± 1.59 µg/ml), nor could it
render native Id-pulsed DCs immunogenic.
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Linkage of xenogeneic carrier proteins such as KLH to weak,
self-derived tumor Ags can enhance their immune recognition
(16). We next asked whether the chemical coupling of KLH
to Id could enhance its immunogenicity when used for DC pulsing. Mice
received three biweekly i.v. injections of DCs pulsed with medium
alone, Id-HCR, Id-HCR-GM, or Id-KLH. Immunization of mice with DCs
pulsed with Id-KLH generated a surprisingly high anti-Id response,
surpassing even that of DCs pulsed with Id-HCR-GM (Fig. 2). Vaccination with DCs pulsed with an
unrelated control IgM/ (4C5) coupled to KLH failed to elicit any
anti-38C13-specific Abs (data not shown). The enhanced
immunogenicity of Id-KLH-pulsed DCs was not simply due to aggregation
of the Id, as the use of Id conjugated to itself for DC pulsing yielded
no anti-Id response. Likewise, concurrent administration of an
equivalent number of DCs pulsed only with KLH along with DCs pulsed
with native Id failed to elicit a response, demonstrating the inability
of carrier-pulsed DCs to supply sufficient helper activity to
coadministered Id-pulsed DCs for stimulation of an anti-Id humoral
response (data not shown).
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To further characterize the anti-Id immune response to
vaccination with DCs pulsed with modified Id proteins, we examined the
isotype profiles of IgG anti-Id Abs following vaccination (Fig. 3). Vaccination of mice with chimeric
Id-HCR-GM fusion protein without DCs induced high levels of anti-Id
Abs exclusively of the IgG1 isotype. In contrast, vaccination with DCs
pulsed with Id-HCR-GM induced lower levels of Abs, but with substantial
proportions of the IgG2a isotype. Similarly, vaccination with Id-KLH
alone led to a primarily IgG1 anti-Id response, while vaccination
with Id-KLH-pulsed DCs led to high titers of anti-Id Abs with a
striking predominance of the IgG2a isotype characteristic of a Th1-type
response (33). These data argue against carryover of
soluble protein Ag by the DCs as the sole mechanism for induction of
the humoral anti-Id response, because the responses to Ag-pulsed
DCs differ qualitatively from those elicited by injection of the same
proteins without DCs.
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Given the superior humoral anti-Id immune response generated
by vaccination with Id-KLH-pulsed DCs, we tested vaccinated mice for
resistance to challenge with 38C13 lymphoma cells (Fig. 4). Mice vaccinated with DCs pulsed with
native Id protein and challenged s.c. with 1000 38C13 tumor cells all
died within 5 wk and were not protected compared with those receiving
PBS. However, a majority of mice vaccinated with Id-KLH-pulsed DCs
survived the tumor challenge (p < 0.0001 and
p < 0.0005 for Id-KLH-pulsed DCs vs PBS and native
Id-pulsed DCs, respectively). Comparable results were obtained in two
additional experiments. No significant protection from tumor challenge
was observed following vaccination with DCs pulsed with Id conjugated
to itself or with an irrelevant IgM/ (4C5) coupled to KLH (data not
shown).
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A). However, vaccination with
Id-KLH-pulsed DCs provided superior protection over Id-KLH plus QS21,
with the majority of animals surviving >60 days
(p = 0.047). Analogous results were obtained in
two additional experiments. Fig. 5B shows the pooled
survival results of the three independent experiments and demonstrates
the consistent advantage of Id-KLH-pulsed DCs over Id-KLH plus chemical
adjuvant in providing tumor protection (p =
0.0082).
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We next sought to determine the contribution of effector T cells
to tumor protection in mice vaccinated with Id-KLH-pulsed DCs. Groups
of mice given three biweekly vaccinations with Id-KLH-pulsed DCs were
treated with mAbs to deplete effector CD4+ or
CD8+ T cells before and following tumor challenge
as described in Materials and Methods (Fig. 6). Depletion of each T cell subset from
the peripheral blood of vaccinated mice was confirmed by flow cytometry
using non-cross-blocking anti-CD4 and anti-CD8 mAbs before
tumor challenge (data not shown). Depletions were maintained throughout
the experiment by continued weekly administration of the depleting
mAbs. The isotype-matched control mAbs used had no significant effect
on the relevant T cell population. Mice depleted of
CD4+ or CD8+ T cells
following vaccination with Id-KLH-pulsed DCs had no statistically
significant alterations in survival compared with mice receiving
isotype-matched control mAbs (p = 0.325 and
p = 0.136, respectively) or compared with those
receiving no mAbs (p = 0.802 and
p = 0.107, respectively).
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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We chose bone marrow-derived DCs cultured in GM-CSF and IL-4 for this
study, as these cells resemble the monocyte-derived DCs most widely
used in clinical trials of DC vaccination for cancer (13, 28). In concordance with our clinical studies (18, 25), vaccination of mice with native Id-pulsed DCs failed to
elicit a humoral anti-Id response (Fig. 1
). However, in our mouse
model, the addition of xenogeneic constant region sequences to the Id
endowed it with the capacity to stimulate an anti-Id humoral
response when used for DC pulsing (Figs. 1 and 2). This was presumably
due to the introduction of foreign helper epitopes, thus creating a
hapten-carrier system (34). The addition of GM-CSF
sequence to this chimeric Id protein further augmented the
immunogenicity of the pulsed DC vaccine, as judged by the humoral
response assay (Figs. 1 and 2). The mechanism of the GM-CSF moiety’s
effect is probably due to stimulation of the Ag uptake or Ag
presentation functions of the DCs (28) rather than to the
contribution of additional helper epitopes, because a chimeric Id
fusion protein containing human GM-CSF (which is not biologically
active in the mouse) does not show enhanced immunogenicity over that of
chimeric Id alone (data not shown). The inability of soluble, unlinked
GM-CSF to boost the immunogenicity of the chimeric Id to that of the
Id-HCR-GM fusion protein may also suggest targeting of the GM-CSF
fusion protein to the DCs by binding to the GM-CSF receptor.
We expected that anti-Id T cell responses would be detectable
following Id-pulsed DC vaccination. Bone marrow-derived DCs cultured in
GM-CSF and IL-4 have been previously shown to induce protective
anti-tumor immunity mediated by CD8+ T cells
when pulsed with the model protein tumor Ags ß-galactosidase and OVA
(6, 8). Indeed, we confirmed that OVA-pulsed DCs could
readily induce cytolytic activity toward OVA-expressing E.G7 cells, yet
under analogous conditions no Id-specific CTL activity could be
generated against 38C13 lymphoma cells (data not shown). In addition,
based upon the in vivo T cell depletion data,
CD4+ or CD8+ T cells appear
to play no significant role as effectors of tumor protection in mice
vaccinated with Id-KLH-pulsed DCs (Fig. 6).
Our findings underscore the important role of DCs in the generation of humoral immune responses. DCs have long been known to be critical for the development of Ab responses to T-dependent Ags by priming Ag-specific Th cells to secrete soluble B cell stimulatory factors (35, 36) and up-regulate CD40 ligand (37). However, a more complex role for DCs in the humoral response was suggested by observations of their clustering with B cells both in vitro (36) and in vivo (38). In some of the earliest studies of Ag-pulsed DC vaccination, injection of mice with splenic DCs pulsed with viral particles or soluble protein Ag was found to stimulate a specific Ab response without the need for additional adjuvants (2, 39, 40). The Abs induced included significant proportions of the IgG2a isotype (2, 40), indicative of a Th1 response (33). These findings suggested the transfer of native, unprocessed Ag to B cells, although the nature of such Ag handling by DCs was not defined. Recently, Wykes et al. demonstrated the transfer of native protein Ag from DCs to B cells to generate a class-switched humoral response (41). Splenic DCs pulsed with DNP-KLH were shown to take up and retain unprocessed Ag in an intracellular compartment for at least 48 h. Induction of DNP-specific IgG required DC to be intact and in direct contact with the responding B cells. Dubois and colleagues (42) have provided further evidence for the direct signaling of DCs to B cells with the demonstration that DCs could stimulate both proliferation and Ab secretion by CD40-activated B cells. These effects are dependent on direct DC-B cell interactions as well as soluble factors, including IL-12 (43). The recent identification of the novel B cell-specific growth and differentiation factor, BAFF, as a product of DCs also confirms the direct influence of DCs over the humoral immune response (44).
A role of DCs as vehicles for the delivery of native, unprocessed Ags
to B cells explains the efficiency of DCs in augmenting the humoral
anti-Id responses observed in our study. As the Id-KLH pulsed DCs
were washed extensively before injection, the amount of Ag delivered
would be expected to be small relative to that of a
protein-plus-adjuvant vaccine, yet the pulsed DCs elicited similar Ab
titers (Fig. 3). Prior reports of humoral response induction by
Ag-pulsed DCs have made use of splenic DCs (2, 35, 36, 39, 40, 41). The present study is the first description of bone
marrow-derived DCs pulsed with tumor Ag eliciting a protective humoral
immune response. Thus, it appears that in vitro-generated DCs can also
serve as efficient vehicles for the delivery of Ag to prime a humoral
immune response.
The mechanisms of protective tumor immunity following anti-Id
vaccination have been proposed to include both Abs (16, 17, 29, 32) and T cell effectors (16, 30, 45, 46, 47, 48) in
various murine lymphoma (16, 17, 29, 30, 32, 47) and
myeloma (45, 46, 48) models. Early investigations into the
protective mechanisms of 38C13 tumor immunity following vaccination
with Id-KLH plus chemical adjuvants suggested a role for
CD4+ and CD8+ T cells based
on in vivo depletion experiments (29). However, immune
serum transfer studies clearly demonstrated that Abs could be
sufficient for tumor protection (29). More recent studies
in the 38C13 lymphoma model using Id-encoding plasmid DNA vaccination
have failed to demonstrate any role for effector T cells in tumor
protection despite the general propensity of DNA vaccination to elicit
T cell immunity and, in particular, CTL (32). In addition,
direct cytotoxicity of Id-immune CTL toward 38C13 tumor cells has never
been demonstrated (29, 30, 32, 47). The present study
provides no exception, as no dependence on T cell effectors was
observed (Fig. 6). Nonetheless, it remains possible that under certain
vaccination conditions, T cell effectors may contribute to immunity
against 38C13 Id (30, 47). In humans, the ability to
elicit anti-Id T cell immunity is likely to depend on an individual
tumor’s Id sequences and the host haplotype. Some individual Ids may
contain sequences capable of eliciting anti-Id
CD4+ and/or CD8+ T cells,
while others may not. This variation may account for the finding of
anti-Id cellular proliferative responses in only 50% of lymphoma
patients vaccinated with DCs pulsed with tumor-derived Id
(25).
Because T cells were not found to mediate tumor protection following
Id-KLH-pulsed DC vaccination in this model, anti-Id Abs are likely
to be necessary effectors. Vaccination using both DCs and the QS21
adjuvant elicited high titers of anti-Id Abs. However, tumor
protection was superior with the use of DCs (Fig. 5). Delivery of Ag by
DCs resulted in a higher proportion of Abs of the IgG2a isotype,
possibly due to DC-derived IL-12 favoring the development of a Th1
response and its associated isotype profile (43, 49, 50).
Higher titers of anti-Id Abs of the IgG2a subclass may contribute
to the greater protection seen with pulsed DC vaccination. In studies
of passive anti-Id mAb therapy for 38C13 lymphoma, anti-Id Abs
of the IgG2a subclass were up to 100-fold more potent in conferring
tumor protection than their class-switched IgG1 counterparts
(51). However, we found no correlation between the serum
anti-Id levels of individual mice and their degree of tumor
protection in the current study (data not shown). This suggests that
another rate-limiting effector function (such as Ab-dependent cellular
cytotoxicity) may have been more efficiently induced by DC
vaccination.
Our findings differ from those of Flamand et al. (2), who
describe the induction of anti-Id Abs and protective immunity
against the BCL-1 murine lymphoma following vaccination with native
BCL-1 Id-pulsed splenic DCs. In contrast, we observed that conjugation
of 38C13 Id protein to KLH was required to elicit both anti-Id Abs
and tumor protection. However, there are several important differences
between these two studies. First, Flamand et al. used splenic DCs,
whereas in the current study bone marrow-derived DCs were used.
Secondly, the BCL-1 and 38C13 variable region sequences share little
homology and may thus differ significantly in their content of helper
epitopes capable of priming CD4+ T cells required
for efficient induction of the humoral anti-Id response.
Furthermore, in the current study we demonstrated that DCs can function
as a superior adjuvant for the induction of protective immunity against
the 38C13 tumor compared with the chemical adjuvant QS21 (Fig. 5),
while Flamand et al. did not demonstrate an advantage of BCL-1-pulsed
DCs over Id protein administered s.c. in CFA.
Despite the expectation that DCs would favor the generation of anti-Id T cell immunity, we have found the tumor-protective effects of Id-KLH-pulsed DC vaccination to be dependent largely, if not exclusively, on the humoral anti-Id response in the 38C13 tumor model. In the case of Id vaccination for B cell lymphomas, induction of a humoral response is desirable given the proven anti-tumor effects of anti-Id Abs (14, 17, 29, 32, 52). Humoral anti-tumor responses may also be advantageous against other cancers that are susceptible to Ab-mediated control. As such, our findings provide rationale for the use of KLH-conjugated Id for DC pulsing in lymphoma clinical trials and suggest a strategy with possible implications for DC vaccination against other human cancers.
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Acknowledgments |
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Footnotes |
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2 Address correspondence and reprint requests to Dr. Ronald Levy, Department of Medicine, Division of Oncology, Stanford University Medical Center, Room M207, Stanford, CA 94305.
3 Abbreviations used in this paper: DC, dendritic cell; Id, B cell lymphoma idiotype; KLH, keyhole limpet hemocyanin; HCR, human constant regions; Id-KLH, Id coupled to KLH.
Received for publication October 28, 1999. Accepted for publication February 16, 2000.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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J. D. Geiger, R. J. Hutchinson, L. F. Hohenkirk, E. A. McKenna, G. A. Yanik, J. E. Levine, A. E. Chang, T. M. Braun, and J. J. Mule Vaccination Of Pediatric Solid Tumor Patients with Tumor Lysate-pulsed Dendritic Cells Can Expand Specific T Cells and Mediate Tumor Regression Cancer Res., December 1, 2001; 61(23): 8513 - 8519. [Abstract] [Full Text] [PDF]
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M. Schnurr, P. Galambos, C. Scholz, F. Then, M. Dauer, S. Endres, and A. Eigler Tumor Cell Lysate-pulsed Human Dendritic Cells Induce a T-Cell Response against Pancreatic Carcinoma Cells: an in Vitro Model for the Assessment of Tumor Vaccines Cancer Res., September 1, 2001; 61(17): 6445 - 6450. [Abstract] [Full Text] [PDF]
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K. Shimizu, E. K. Thomas, M. Giedlin, and J. J. Mulé Enhancement of Tumor Lysate- and Peptide-pulsed Dendritic Cell-based Vaccines by the Addition of Foreign Helper Protein Cancer Res., March 1, 2001; 61(6): 2618 - 2624. [Abstract] [Full Text]
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J. M. Timmerman, C. B. Caspar, S. L. Lambert, A. D. Syrengelas, and R. Levy Idiotype-encoding recombinant adenoviruses provide protective immunity against murine B-cell lymphomas Blood, March 1, 2001; 97(5): 1370 - 1377. [Abstract] [Full Text] [PDF]
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K. A. Candido, K. Shimizu, J. C. McLaughlin, R. Kunkel, J. A. Fuller, B. G. Redman, E. K. Thomas, B. J. Nickoloff, and J. J. Mulé Local Administration of Dendritic Cells Inhibits Established Breast Tumor Growth: Implications for Apoptosis-inducing Agents Cancer Res., January 1, 2001; 61(1): 228 - 236. [Abstract] [Full Text]
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 O. W. Press, J. P. Leonard, B. Coiffier, R. Levy, and J. Timmerman Immunotherapy of Non-Hodgkin's Lymphomas Hematology, January 1, 2001; 2001(1): 221 - 240. [Abstract] [Full Text] [PDF]
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