An autologous leukemia cell vaccine prevents

From www.bloodjournal.org by guest on February 4, 2015. For personal use only.
Blood First Edition Paper, prepublished online September 18, 2014; DOI 10.1182/blood-2014-04-568956
GIBBINS et al
IMMUNOTHERAPY
CHEMOTHERAPY ENABLES POTENT LEUKEMIA
1
An autologous leukemia cell vaccine prevents murine acute
2
leukemia relapse after cytarabine treatment
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
John D. Gibbins1,2, Lindsay R. Ancelet1, Robert Weinkove1,3,4, Benjamin. J
Compton5, Gavin F. Painter5, Troels R. Petersen1 and Ian F. Hermans1,2
1
Malaghan Institute of Medical Research, PO Box 7060, Wellington 6242,
New Zealand
2
School of Biological Sciences, Victoria University of Wellington, PO Box 600
Wellington 6140, New Zealand
3
Capital and Coast District Health Board, Wellington Hospital, Wellington
6021, New Zealand
4
Department of Pathology and Molecular Medicine, University of Otago,
Wellington, PO Box 7343, Wellington 6242, New Zealand
5
Ferrier Research Institute, Victoria University of Wellington, PO Box 33-436,
Petone 5046, New Zealand
Correspondence: Ian F. Hermans, Malaghan Institute of Medical Research,
PO Box 7060, Wellington 6242, New Zealand. Phone: +6444996914; Fax:
+6444996915; e-mail: [email protected]
Short title: Chemotherapy enables potent leukemia immunotherapy
Text word count: 4510
Abstract word count: 196
Number of figures: 7
Number of references: 75
Scientific category: Immunobiology
Submitted: April 9, 2014
30
31
32
Key points
1
Copyright © 2014 American Society of Hematology
From www.bloodjournal.org by guest on February 4, 2015. For personal use only.
GIBBINS et al
IMMUNOTHERAPY
CHEMOTHERAPY ENABLES POTENT LEUKEMIA
1
A cellular vaccine incorporating the glycolipid α-galactosylceramide prevents
2
relapse of acute leukemia following cytarabine chemotherapy
3
2
From www.bloodjournal.org by guest on February 4, 2015. For personal use only.
GIBBINS et al
IMMUNOTHERAPY
CHEMOTHERAPY ENABLES POTENT LEUKEMIA
1
Abstract
2
Acute leukemias with adverse prognostic features carry a high relapse rate
3
without allogeneic stem cell transplantation (allo-SCT). Allo-SCT has a high
4
morbidity and is precluded for many patients due to advanced age or
5
comorbidities. Post-remission therapies with reduced toxicities are urgently
6
needed. The murine acute leukemia model C1498 was used to study the
7
efficacy of an intravenously administered vaccine consisting of irradiated
8
leukemia cells loaded with the natural killer T (NKT) cell agonist α-
9
galactosylceramide (α-GalCer). Prophylactically, the vaccine was highly
10
effective at preventing leukemia development through the downstream
11
activities of activated NKT cells, which was dependent on splenic langerin+
12
CD8α+ dendritic cells and lead to stimulation of anti-leukemia CD4+ and CD8+
13
T cells. However, hosts with established leukemia received no protective
14
benefit from the vaccine, despite inducing NKT cell activation. Established
15
leukemia was associated with increases in regulatory T cells and myeloid-
16
derived suppressor cells, and the leukemic cells themselves were highly
17
suppressive in vitro. Although this suppressive environment impaired both
18
effector arms of the immune response, CD4+ T cell responses were more
19
severely affected. When cytarabine chemotherapy was administered prior to
20
vaccination, all animals in remission post-therapy were protected against
21
rechallenge with viable leukemia cells.
22
3
From www.bloodjournal.org by guest on February 4, 2015. For personal use only.
GIBBINS et al
IMMUNOTHERAPY
CHEMOTHERAPY ENABLES POTENT LEUKEMIA
1
Introduction
2
Induction chemotherapies for acute leukemias typically induce morphologic
3
remission, but without allogeneic stem cell transplantation (allo-SCT), most
4
patients with high-risk genetic features subsequently relapse.1-5 Allo-SCT has
5
a high morbidity and mortality, is costly, and is often precluded by age, co-
6
morbidities or lack of a suitable donor.6-8 There is an unmet need for effective
7
post-remission therapies that do not carry the toxicities and cost of allo-SCT.9
8
9
Relapse of acute leukemia is mediated by a population of blasts that fall
10
below the threshold used to define morphologic remission, but may be
11
detected using sensitive flow cytometric or molecular assays.10-14 In addition
12
to over expressing certain self-antigens,15 leukemic blasts harbor numerous
13
mutations,16 resulting in expression of tumor-specific antigens capable of
14
eliciting autologous CD4+ and CD8+ T cell responses.17 This can potentially be
15
exploited by post-remission immunotherapy.18,19
16
17
The use of irradiated whole leukemia cells in vaccines for post-remission
18
immunotherapy is technically feasible,20 and has the potential to elicit immune
19
responses against multiple leukemia-specific antigens without needing to first
20
define leukemia-specific T cell epitopes or patient tissue type. However,
21
administration of a vaccine without a suitable adjuvant is unlikely to elicit an
22
effective immune response and may lead to tolerance.21,22 The glycolipid α-
23
galactosylceramide (α-GalCer) has recently been shown to be a useful
24
adjuvant for whole tumor cell vaccination by eliciting stimulatory interactions
25
between dendritic cells (DCs) and natural killer T (NKT) cells.23-26 When DCs
4
From www.bloodjournal.org by guest on February 4, 2015. For personal use only.
GIBBINS et al
IMMUNOTHERAPY
CHEMOTHERAPY ENABLES POTENT LEUKEMIA
1
acquire cellular material from irradiated tumor cells that have been treated
2
with α-GalCer, the protein content is presented as peptides via MHC
3
molecules to CD4+ and CD8+ T cells, while the α-GalCer is presented via the
4
MHC-like molecule CD1d to NKT cells. Interactions between DCs and NKT
5
cells promote CD40 signaling, leading to DC activation, which increases their
6
capacity to stimulate peptide-specific T cells.23,24 Significantly, vaccines
7
comprised of irradiated tumor cells pulsed with α-GalCer have been shown to
8
be effective in murine models of hematopoietic malignancies, including acute
9
myeloid leukemia (AML) and acute lymphoid leukemia (ALL),23,27,28 and in
10
other malignancies.25,27,29
11
12
Cancer-associated immunosuppression can present a significant barrier to
13
effective vaccine-based immunotherapy.30 AML generates an
14
immunosuppressive environment,31,32 characterised by impaired DC function33
15
and increased levels of regulatory T cells (Tregs).35-37 It follows that
16
immunotherapy may be most effectively used during morphologic remission
17
after induction chemotherapy.
18
19
Here we investigated the efficacy of a vaccine comprised of irradiated
20
leukemia cells pulsed with α-GalCer in a murine acute leukemia model. While
21
vaccination was capable of eliciting a leukemia-specific T cell response in
22
mice with established disease, the activity was impaired by leukemia-
23
associated immunosuppression. However, when the vaccine was
24
administered to mice in remission after cytarabine chemotherapy, it protected
25
against rechallenge with an increased dose of viable leukemic blasts. These
5
From www.bloodjournal.org by guest on February 4, 2015. For personal use only.
GIBBINS et al
IMMUNOTHERAPY
CHEMOTHERAPY ENABLES POTENT LEUKEMIA
1
findings have implications for the design of clinical trials testing
2
immunotherapies for acute leukemias.
3
6
From www.bloodjournal.org by guest on February 4, 2015. For personal use only.
GIBBINS et al
IMMUNOTHERAPY
1
CHEMOTHERAPY ENABLES POTENT LEUKEMIA
Materials and Methods
2
3
Animal ethics
4
Inbred C57BL/6 mice were purchased from Jackson Laboratories, Bar Harbor,
5
Maine. Also used were: lang-EGFPDTR and lang-EGFP mice, which express
6
the human diphtheria toxin (DT) receptor and/or enhanced green fluorescent
7
protein (EGFP) under the langerin promoter;38 and FoxP3-GFP mice, that
8
have EGFP inserted into the first coding exon of the Foxp3 gene.39 All animals
9
were bred and housed at the Malaghan Institute of Medical Research
10
Biomedical Research Unit, Wellington, New Zealand. Experiments were
11
approved by the Animal Ethics Committee, Victoria University, Wellington,
12
New Zealand; reference 2012R28M.
13
14
Media and reagents
15
The acute leukemia line C1498,40 (ATCC, Manassas, VA, USA), was cultured
16
in Iscove’s Modified Dulbecco’s Medium (IMDM) supplemented with 5% fetal
17
bovine serum (FBS) (SAFC Bioscience, Auckland, New Zealand), 100 U/mL
18
penicillin/100 g/mL streptomycin, 50 M 2-mercaptoethanol (all from Invitrogen,
19
Auckland, New Zealand). α-GalCer was manufactured by synthesizing a
20
protected phytosphingosine derivative from phytosphingosine (TCI, P1765) as
21
previously described. 41.42
22
23
Leukemia challenge treatment with whole tumor vaccines
24
For leukemia challenge experiments, mice were administered 1 x 105 C1498
25
cells intravenously via the lateral tail vein, unless otherwise stated. To
7
From www.bloodjournal.org by guest on February 4, 2015. For personal use only.
GIBBINS et al
IMMUNOTHERAPY
CHEMOTHERAPY ENABLES POTENT LEUKEMIA
1
generate vaccines, C1498 cells were cultured for 24 hours in IMDM with 200
2
ng/ml of α-GalCer, washed with phosphate-buffered saline (PBS), and γ-
3
irradiated to 150 Gy. Vaccines comprised of of 7.5 x 105 cells were
4
administered intravenously. Mice were monitored for onset of leukemia-
5
associated symptoms, such as weight loss or overt behavioral symptoms
6
(hunching, reduced activity or reduced grooming) and were euthanized
7
following symptom development. Although weight loss was monitored for all
8
mice in symptom-free survival experiments, the onset of symptoms often
9
preceded weight loss in this model. Experiments were conducted with 5-6
10
animals per treatment group. CD4+ or CD8+ T cells were depleted by injection
11
of anti-CD4 antibodies (GK1.5; 125 μg per mouse), or anti-CD8 antibodies
12
(2.43; 250 μg per mouse), respectively, administered five, twelve and
13
nineteen days following vaccination; depletion methods were sufficient to
14
maintain >95% depletion for GK1.5 and >90% for 2.43 over the course of the
15
experiment (Supplementary Figure 1). Anti-CD25 (clone PC61) was used to
16
deplete Tregs, resulting in >95% reduction of CD4+ CD25+ cells
17
(Supplementary Figure 2). Langerin+ DCs were depleted from Lang-
18
EGFPDTR mice by intraperitoneal administration of 350 ng of DT two days
19
before vaccine, resulting in >95% reduction of langerin+ CD8α+ DCs for three
20
days.43,44 In some experiments three doses of 3 mg of cytarbine (Pfizer,
21
Auckland, New Zealand) was administered ten hours apart the day following
22
leukemia challenge.
23
24
Histology
8
From www.bloodjournal.org by guest on February 4, 2015. For personal use only.
GIBBINS et al
IMMUNOTHERAPY
CHEMOTHERAPY ENABLES POTENT LEUKEMIA
1
Femurs were placed in 4% formalin (Sigma-Aldrich, MO, U.S.A), decalcified
2
with 10% formic acid and processed. Paraffin embedded sections were
3
stained with haematoxylin-and-eosin (made in house) and blood smears were
4
stained with Romanowsky stain variant (Siemens Healthcare, Erlangen,
5
Germany). Slides were examined with an Olympus BX51 microscope
6
(Precision Microscopy Equipment Wellington, New Zealand) and captured
7
with an Olympus DP70 (Precision Microscopy Equipment, Wellington, New
8
Zealand) using Cell^F software (Olympus).
9
10
Cytokine production assay
11
Supernatant cytokine levels were measured by cytokine bead array (Biorad
12
Laboratories, Inc, Auckland, New Zealand) following culture with one-million
13
splenocytes and ten-thousand bone marrow-derived DCs (BM-DC) loaded
14
with C1498 lysate for 4 hours. BM-DCs were prepared from syngeneic bone
15
marrow cultured in IL-4 and GM-CSF for six days, followed by 18 hours with
16
100 ng/ml of LPS; lysate from C1498 was added at a ratio of one DC to the
17
equivalent of six tumor cells for the last four hours.
18
19
T cell suppression assay
20
Lymph node preparations from naïve C57BL/6 were stained with
21
carboxyfluorescein succinimidyl ester (CFSE), and cultured for 72 hours with
22
2 µg/mL anti-CD3 (clone 2C11) and 2 µg/mL anti-CD28 (clone 37.51) (both
23
prepared in house) in the presence of purified splenic CD11b+ cells or C1498
24
cells. CFSE dilution on T cells was analyzed by flow cytometry.
25
9
From www.bloodjournal.org by guest on February 4, 2015. For personal use only.
GIBBINS et al
IMMUNOTHERAPY
CHEMOTHERAPY ENABLES POTENT LEUKEMIA
1
Flow cytometry
2
Nonspecific FcR binding was blocked with anti-CD16/32 clone 2.4G2
3
(prepared in house). Dead cells were excluded by staining with propidium
4
iodide (BD Pharmingen, California, USA), 4, 6-diamidino-2-phenylindole
5
(DAPI) (Invitrogen) or LIVE/DEAD® Fixable Blue (Invitrogen). For intracellular
6
staining, cells were restimulated for 20 hours with anti-CD3 and anti-CD28
7
antibodies and 1 µg/ml of monensin was added for the last four hours of
8
incubation. Flow cytometry was performed using a FACSCalibur or LSRII (BD
9
Biosciences) and analyzed using FlowJo software (TreeStar Inc.). Doublet
10
and dead cell exclusion was performed. The antibodies used were: anti-CD3-
11
FITC (145-2C11), CD3-PE-Cy7 (145-2C11) anti-CD4-A488 (GK1.5), anti-
12
CD8-A700 (53-6.7), anti-CD11b biotin (M170), anti-CD86 PE (GL-1) anti-
13
CD44-PerCP-Cy5.5 (IM7), anti-FoxP3-PE (FJK-16s) and anti-IFN-γ-PE-Cy7
14
(XMG1-2), all from eBioscience, Auckland, New Zealand; anti-CD11c-PE-Cy7
15
(N418), anti-CD4-APC (GK1.5), anti-CD8-FITC (53-6.7) anti-CD11c-APC
16
(N418), all from Biolegend, San Diego, USA; anti-CD8-Pacific blue (53-6.7)
17
anti-CD40-biotinylated (3/23), streptavidin-PE-Cy7, all from BD Bioscience.
18
Invariant NKT cells were detected using α-GalCer-loaded CD1d tetramers
19
(NIH Tetramer Core Facility, Atlanta, USA).
20
21
Statistical analyses
22
Bars and error bars depict the mean and standard deviation of the mean. For
23
comparisons of one variable, the Mann–Whitney test was used for unpaired
24
data, the Wilcoxon-matched pairs test for paired data, and a one-way analysis
25
of variance (ANOVA) with a Bonferroni post-test for experiments comparing
10
From www.bloodjournal.org by guest on February 4, 2015. For personal use only.
GIBBINS et al
IMMUNOTHERAPY
CHEMOTHERAPY ENABLES POTENT LEUKEMIA
1
more than two groups. The log-rank test was used to determine significance
2
between Kaplan Meier survival curves. Analysis was performed with Prism 5.0
3
software (GraphPad Software, Inc.); P values of <0.05 were considered
4
significant.
11
From www.bloodjournal.org by guest on February 4, 2015. For personal use only.
GIBBINS et al
IMMUNOTHERAPY
1
CHEMOTHERAPY ENABLES POTENT LEUKEMIA
Results
2
3
A vaccine comprised of irradiated α-GalCer-pulsed leukemia cells
4
protects against acute leukemia through the activities of CD4+ and CD8+
5
T cells and langerin+ CD8α+ DCs
6
Mice intravenously challenged with the cell line C1498 developed leukemia,
7
characterized by replacement of normal bone marrow hematopoiesis and
8
leukocytosis with circulating blasts (Figure 1A-C). However, mice vaccinated
9
with irradiated leukemia cells pulsed with the glycolipid adjuvant α-GalCer
10
(leukemia/α-GalCer) seven days before C1498 challenge did not develop
11
leukocytosis (Figure 1C) and no blasts were seen in the peripheral blood
12
smear or by histologic examination of bone marrow (Figure 1A-C). Animals
13
vaccinated with the leukemia/α-GalCer vaccine were protected from leukemia
14
development and remained symptom-free for the duration of the experiment
15
(Figure 1D). Vaccination with irradiated leukemia cells without α-GalCer, or
16
with α-GalCer alone, did not protect hosts from leukemia development (Figure
17
1D).
18
19
To determine the effector cells responsible for vaccine-induced protection
20
against leukemia development, mice were vaccinated and depleted of CD4+
21
or CD8+ cells two days before C1498 challenge to ensure that the depletion
22
would not interfere with cells potentially important for immune priming.
23
Vaccine-induced protection was reduced following depletion of either CD4+ or
24
CD8+ cells, suggesting that both CD4+ and CD8+ T cells mediated the
25
protection afforded by the vaccine (Figure 1E).
12
From www.bloodjournal.org by guest on February 4, 2015. For personal use only.
GIBBINS et al
IMMUNOTHERAPY
CHEMOTHERAPY ENABLES POTENT LEUKEMIA
1
2
In the spleen, CD8α+ DCs are thought to be responsible for phagocytosing
3
circulating apoptotic cells,21 and a subpopulation of these cells in the marginal
4
zone that express CD103 and langerin have been shown to induce robust
5
cytotoxic T cell responses through efficient CD8+ T cell cross-priming.44-46 To
6
establish whether langerin+ DCs were involved in the protection afforded by
7
the leukemia/α-GalCer vaccine, lang-EGFPDTR hosts were depleted of
8
langerin-expressing cells by DT treatment before vaccination as previously
9
described.44 This led to complete abrogation of the protective effect of
10
leukemia/α-GalCer (Figure 1F), indicating that langerin+ DCs are essential for
11
vaccine efficacy.
12
13
Vaccination is ineffective in the presence of established leukemia
14
despite retaining capacity to stimulate NKT cells and DCs
15
Having shown that prophylactic leukemia/α-GalCer vaccination can elicit an
16
anti-tumor effect, we next determined whether the vaccine could prolong
17
survival in mice with established leukemia. Mice inoculated with C1498 one
18
week before vaccination with leukemia/α-GalCer received no therapeutic
19
benefit, and the onset of symptoms was comparable to non-vaccinated
20
controls (Figure 2A).
21
22
To explore why therapeutic vaccination of mice with established leukemia was
23
ineffective, we investigated the cascade of immune activation involved in α-
24
GalCer-adjuvanted vaccination. Since the adjuvant effect of α-GalCer involves
25
reciprocal interaction and activation of NKT cells and DCs,47-50 we first
13
From www.bloodjournal.org by guest on February 4, 2015. For personal use only.
GIBBINS et al
IMMUNOTHERAPY
CHEMOTHERAPY ENABLES POTENT LEUKEMIA
1
assessed the level of NKT cell activation by analyzing NKT cell number and
2
function in animals vaccinated seven or fourteen days after C1498 challenge.
3
A substantial increase in the number of splenic NKT cells was observed in all
4
vaccinated animals compared with unvaccinated controls, although there was
5
a trend towards reduced accumulation of NKT cells in leukemia-bearing
6
animals (Figure 2B,C). A notable feature of activated NKT cells is the ability to
7
rapidly produce high levels of the cytokines IL-4 and IFN-γ, which can be
8
detected in serum.47 Both cytokines were detected in host serum after
9
vaccination and levels were similar in mice with and without established
10
leukemia (Figure 2D-E). Together these data suggest that the capacity of the
11
vaccine to stimulate NKT cells in established leukemia is largely intact.
12
13
We next determined whether the presence of established leukemia impaired
14
the ability of the vaccine to activate DCs. Lang-EGFP hosts were used to
15
identify langerin+ CD8α+ DCs by flow cytometry. A decrease in the proportion
16
of the langerin+ CD8α+ subset of DCs was found in the spleens of all
17
vaccinated mice regardless of presence of established leukemia, which is
18
consistent with previous reports showing these DCs are depleted in response
19
to NKT cell stimulation (Figure 2F and G).21,45,51 The remaining langerin+
20
CD8α+ DCs upregulated CD40 and CD86 after vaccination, irrespective of the
21
presence of established leukemia (Figure 2H and I), although the expression
22
of CD86 on langerin+ CD8α+ DCs was reduced in mice with established
23
leukemia. The expression of CD40 and CD86 was similarly upregulated in
24
CD8α+ DCs (Figure 2J and K).
25
14
From www.bloodjournal.org by guest on February 4, 2015. For personal use only.
GIBBINS et al
IMMUNOTHERAPY
CHEMOTHERAPY ENABLES POTENT LEUKEMIA
1
The interaction between DCs and NKT cells involves CD40/CD40 ligand
2
interaction that contributes to significant production of IL-12, primarily from
3
langerin+ CD8α+ DCs.44 Serum IL-12 was analyzed five hours after vaccine
4
administration in the presence or absence of established leukemia, and
5
similar levels were observed (Figure 2L). Together, these results indicate that
6
the inefficacy of the vaccine was not attributed to a failure to activate NKT
7
cells or DCs.
8
9
Vaccine-induced activation of CD4+ T cells is suppressed in the
10
presence of established leukemia
11
To establish whether leukemia suppressed effector T cells, the vaccine-
12
induced T cell response was examined. Splenocytes from animals vaccinated
13
in the presence or absence of established leukemia were cultured with bone
14
marrow-derived DCs loaded with C1498 lysate and levels of IFN-γ in the
15
supernatant were quantified. Splenocytes from vaccinated animals had
16
elevated supernatant IFN-γ following restimulation, suggesting a leukemia
17
antigen-specific immune response had been induced, although overall levels
18
from leukemia-bearing animals were not significantly different from animals
19
without leukemia (Figure 3A). Flow cytometric analysis of splenic CD8+ T cells
20
seven days after vaccination showed that upregulation of the activation
21
marker CD44 and intracellular IFN-γ production were not significantly impaired
22
by the presence of established leukemia (Figure 3C-D). However, established
23
leukemia prevented vaccine-induced CD44 and IFN-γ expression on CD4+ T
24
cells (Figure 3E-F). Therefore, in the established leukemia setting, the
25
induction of leukemia-specific CD4+ T cells was impaired.
15
From www.bloodjournal.org by guest on February 4, 2015. For personal use only.
GIBBINS et al
IMMUNOTHERAPY
CHEMOTHERAPY ENABLES POTENT LEUKEMIA
1
2
Established leukemia is associated with numerous suppressive
3
mechanisms
4
Increased numbers of Tregs are found in patients with acute leukemias, such
5
as AML and ALL.52-57 To determine the involvement of regulatory cells in
6
C1498, the percentage of CD4+ FoxP3+ Tregs was determined by flow
7
cytometry. Mice with established leukemia had a significantly increased
8
percentage of CD4+ FoxP3+ cells in both the liver and spleen (Figure 4A-B).
9
To determine whether alleviation of Treg-related immunosuppression could
10
enhance the therapeutic effect of the leukemia/α-GalCer vaccine, an anti-
11
CD25 antibody was used to deplete Tregs from leukemia-bearing mice before
12
vaccination.58 Mice depleted of Tregs prior to vaccination had increased
13
survival, although all animals ultimately succumbed to leukemia outgrowth
14
(Figure 4C). This was dependent on the vaccine because Treg depletion
15
alone did not delay symptom onset. These results indicate that Tregs are
16
partially responsible for the inefficacy of therapeutic leukemia/α-GalCer
17
vaccination.
18
19
Since myeloid-derived suppressor cells (MDSC) can also potentially
20
contribute to immunosuppression in patients with acute leukemias,52-56 the
21
proportion of MDSCs was assessed in the spleens of leukemic animals. A
22
significantly increased percentage of CD11b+ Ly6G+ cells was observed in
23
animals with established leukemia (Figure 4D-E). These cells could be
24
distinguished from leukemic blasts by CD11b, which is not expressed in
25
C1498 (J.D.G, unpublished data, January 14, 2013). When splenic CD11b+
16
From www.bloodjournal.org by guest on February 4, 2015. For personal use only.
GIBBINS et al
IMMUNOTHERAPY
CHEMOTHERAPY ENABLES POTENT LEUKEMIA
1
cells were isolated 20 days after leukemia challenge and cultured with CFSE-
2
labeled lymphocytes, the proliferation of CD4+ T cells was significantly
3
reduced relative to cultures containing CD11b+ cells from control mice,
4
suggesting greater suppressive activity on a per cell basis (Figure 4F).
5
Although a trend towards reduced CD8+ T cell proliferation was also observed,
6
this failed to reach significance in all experiments (Figure 4G) (J.D.G,
7
unpublished data, January 14, 2013).
8
9
To analyze whether the C1498 line itself had immunosuppressive capabilities,
10
C1498 cells were cultured with CFSE-labeled lymphocytes and T cell
11
proliferation was compared to cultures containing naïve CD11b+ splenocytes.
12
Proliferation of CD4+ and CD8+ T cells was severely impaired by co-culture
13
with C1498 cells, although suppression of CD4+ T cell proliferation was more
14
pronounced (Figure 4H-I). Overall, this data indicates that there may be
15
several immunosuppressive activities at play in the context of established
16
leukemia.
17
18
Cytarabine pretreatment does not suppress vaccine-induced responses
19
While induction chemotherapy can drastically reduce the tumor cell burden in
20
leukemic patients, potentially providing an opportunity for immunotherapeutic
21
intervention, it can also induce lymphopenia and an expansion of Tregs.59 To
22
determine whether cytarabine chemotherapy could be used successfully in
23
combination with immunotherapy, the effect of cytarabine treatment on T cells
24
was analyzed in mice with acute leukemia. While the percentage of T cells
25
was reduced in the spleens of mice with untreated leukemia, mice treated with
17
From www.bloodjournal.org by guest on February 4, 2015. For personal use only.
GIBBINS et al
IMMUNOTHERAPY
CHEMOTHERAPY ENABLES POTENT LEUKEMIA
1
cytarabine after leukemia challenge had similar proportions of CD4+ and CD8+
2
T cells compared to naïve controls (Figure 5A-B). Moreover, while expression
3
of the activation marker CD44 on CD8+ and CD4+ T cells was reduced in
4
animals with leukemia, in cytarabine-treated animals expression was similar
5
to that seen in naïve healthy controls (Figure 5C-D), surprisingly suggesting
6
that rather than suppressing endogenous immune responses, cytarabine may
7
be instead restoring the T cell compartment.
8
9
We next wanted to determine the effect of cytarabine pretreatment on the
10
vaccine-induced response. C1498-challenged mice were treated with
11
cytarabine and 20 days later administered the leukemia/α-GalCer vaccine, or
12
left unvaccinated, and the proportions of Tregs and MDSCs was assessed
13
one week following vaccination. Although we observed a reduction in the
14
percentages of Tregs in both the spleen and liver of vaccinated animals
15
compared to untreated leukemic mice, the addition of cytararbine to the
16
treatment regime had no effect (Figure 6A-B). Similarly, there were no
17
changes in the proportions of MDSCs (Figure 6C-D). The ratio of effector
18
CD8+ T cells (CD44hi) to Tregs was increased in both the spleen and liver of
19
vaccinated mice relative to untreated animals and was also unaffected by
20
cytarabine pretreatment. We observed a similar trend in the ratio of effector
21
CD4+ T cells (CD44hi) to Tregs, again establishing that the addition of
22
cytarabine did not impact the vaccine-induced response in leukemic mice
23
(Figure E-H).
24
18
From www.bloodjournal.org by guest on February 4, 2015. For personal use only.
GIBBINS et al
IMMUNOTHERAPY
CHEMOTHERAPY ENABLES POTENT LEUKEMIA
1
The different treatments were associated with significant changes in
2
proportions of T cells in the bone marrow, the major site of leukemia cell
3
accumulation. Vaccinated animals had an increased proportion of CD3+ T
4
cells compared to unvaccinated mice that was unaffected by cytarbine (Figure
5
6I). Interestingly, leukemia challenge was associated with an increase in the
6
percentage of CD8+ T cells expressing the T cell exhaustion marker
7
programmed-death 1 (PD-1) in the bone marrow, which was unchanged
8
following cytarabine treatment alone. However, treatment with cytabarine
9
followed by vaccination resulted in reduced percentages of PD-1-expressing
10
CD8+ T cells compared to untreated C1498-challenged animals, similar to that
11
observed in vaccinated, non-leukemic mice (Figure 6J). Similarly, the lowest
12
percentage of PD-1 expressing CD4+ T cells was observed in mice that
13
received the combination therapy (6K). These results demonstrate that
14
cytarabine pretreatment does not suppress vaccine-induced immune
15
responses and suggests rather, that the therapies may be successfully
16
combined. Importantly, chemotherapy to induce minimal residual disease for
17
acute leukemia may provide a window for immunotherapy.
18
19
20
Vaccination following cytarabine treatment protects against leukemia
21
rechallenge
22
Since cytarbine pretreatment did not negatively impact vaccine-induced
23
immune responses, we next assessed the efficacy of the leukemia/α-GalCer
24
vaccine as a post-remission therapy (Figure 7A). Mice were challenged with
25
C1498 and then treated with cytarabine. After 20 days, one group was
19
From www.bloodjournal.org by guest on February 4, 2015. For personal use only.
GIBBINS et al
IMMUNOTHERAPY
CHEMOTHERAPY ENABLES POTENT LEUKEMIA
1
vaccinated with the leukemia/α-GalCer vaccine. Cytarabine treatment
2
prolonged survival in both groups after C1498 challenge (Figure 7B) as
3
previously reported,61,62 although all animals eventually relapsed
4
(Supplementary Figure 3). Surviving cytarabine treated animals, as well as
5
additional naïve controls, were challenged with an elevated dose of viable
6
C1498 cells to determine whether they had developed protective immunity to
7
acute leukemia. Strikingly, animals that received vaccination after cytarabine
8
chemotherapy had superior protection from C1498 rechallenge, whereas all
9
animals that received chemotherapy alone developed symptoms associated
10
with leukemia progression within 20 days (Figure 7B). Therefore, vaccination
11
provided durable protection against leukemia when administered during
12
remission following cytarabine chemotherapy, suggesting that an α-GalCer-
13
pulsed irradiated leukemia cell vaccine may be a promising post-remission
14
immunotherapy for acute leukemia.
15
16
17
18
19
Discussion
20
There is an unmet need for effective post-remission therapies for acute
21
leukemia that have reduced toxicity and cost compared to allo-SCT. Using the
22
aggressive acute leukemia cell line C1498,63 we show that a simple vaccine
23
comprised of whole irradiated leukemia cells pulsed with the glycolipid
24
adjuvant α-GalCer protected against leukemia development in vivo. Despite
20
From www.bloodjournal.org by guest on February 4, 2015. For personal use only.
GIBBINS et al
IMMUNOTHERAPY
CHEMOTHERAPY ENABLES POTENT LEUKEMIA
1
displaying immunologic activity, the vaccine did not delay disease progression
2
in mice with established disease, at least in part due to leukemia-related
3
immunosuppressive activities. However, in animals that were in remission
4
following cytarabine chemotherapy, the vaccine protected against leukemia
5
rechallenge.
6
7
The glycolipid α-GalCer acts as an adjuvant via a third-party mechanism by
8
binding to CD1d on DCs and recruiting NKT cells to create a stimulatory
9
environment that leads to enhanced peptide-specific responses by
10
conventional CD4+ and CD8+ effector T cells.23,47,64 While CD1d is weakly
11
expressed on C1498 and has been identified in other acute leukemic cell lines,
12
including AML-ETO9a 23, EL4, and WEHI-3B (L.R.A, unpublished data, June
13
14, 2014), we have previously demonstrated that a CD1d-negative α-GalCer-
14
pulsed glioma vaccine can provide protection against glioma challenge. 25 We
15
have also shown that CD1d-deficient DCs can transfer α-GalCer to host
16
resident CD1d-expressing antigen-presenting cells in vivo to induce potent
17
iNKT cell activation, which likely reflects transfer of α-GalCer embedded
18
within membranes of the injected cells.46 It is therefore not necessary for NKT
19
cells to interact directly via CD1d on the cells of the vaccine to provide
20
adjuvant activity. Rather we favor the hypothesis that host DCs acquire α-
21
GalCer from the leukemia cells of the vaccine; these DCs then become
22
licensed by presenting the α-GalCer via CD1d to NKT cells, in turn leading to
23
enhanced induction of peptide-specific CD4+ and CD8+ effector T cells.
24
Consistent with this mechanism, the activity of the leukemia/α-GalCer vaccine
21
From www.bloodjournal.org by guest on February 4, 2015. For personal use only.
GIBBINS et al
IMMUNOTHERAPY
CHEMOTHERAPY ENABLES POTENT LEUKEMIA
1
required langerin-expressing DCs, and involved CD4+ and CD8+ T cells for full
2
efficacy.
3
4
Therapeutic administration of the leukemia/α-GalCer vaccine was unable to
5
prolong survival in mice with established leukemia, despite stimulating NKT
6
cells, and leading to activation of DCs. We identified several mechanisms by
7
which C1498 could suppress immune responses, including increased
8
proportions of Tregs and MDSCs, and a direct suppressive effect of the
9
leukemia cells on T cell proliferation. It is notable that acute leukemias have
10
been reported to produce arginase65 and indolamine 2,3-dioxygenase66 which
11
may be responsible for this direct effect. Our results suggest that leukemia-
12
specific CD4+ T cells play a significant role in the efficacy of the leukemia/α-
13
GalCer vaccine and depletion experiments indicated that CD4+ T cells were of
14
similar importance as CD8+ T cells for vaccine efficacy. In leukemia-bearing
15
mice, IFN-γ production by CD4+, but not CD8+ T cells was impaired.
16
Interestingly, MDSCs from leukemic mice induced a more pronounced
17
suppression of CD4+ T cells compared to those harvested from non-leukemic
18
animals, although we were unable to show a similar reproducible effect on
19
CD8+ T cells. Also, the potent suppression of T cell proliferation by the
20
leukemia cells themselves appeared to be greater on CD4+ T cells than on
21
CD8+ T cells. It is possible therefore that the leukemic environment induces
22
broad CD4+ T cell dysfunction, perhaps including the inability to provide T cell
23
help to CD8+ T cells. In this context, it is notable that vaccine efficacy in the
24
prophylactic setting was dependent on langerin+ CD8α+ DCs, which have
25
been shown to have a potent capacity for stimulating cytotoxic T cells; the
22
From www.bloodjournal.org by guest on February 4, 2015. For personal use only.
GIBBINS et al
IMMUNOTHERAPY
CHEMOTHERAPY ENABLES POTENT LEUKEMIA
1
vaccine may ultimately depend on CD4+ T cell help to mobilize effective
2
cytotoxic T cells through these DCs. However, given that some anti-tumor
3
activity was seen in the absence of CD8+ T cells, other CD4+ T cell functions
4
must be involved, perhaps as effectors in their own right,67 or through
5
interactions with other MHC class II-expressing cells.
6
7
Other groups have also shown that α-GalCer pulsed acute leukemia cells can
8
be used as a prophylactic vaccine. Using the AML cell line AML-ETO9a,
9
Mattarollo et al23 showed that irradiated α-GalCer-pulsed AML cells prevented
10
AML development in the prophylactic setting but only delayed progression
11
when administered therapeutically, and Shimizu et al29 demonstrated effective
12
prophylactic vaccination using the myelomonocytic WEHI3B model. Our
13
experiments extend these findings by elucidating immunosuppressive
14
activities in established leukemia, and indicating that protection against acute
15
leukemia can also be invoked during remission after cytarabine chemotherapy.
16
As this chemotherapeutic agent is in routine clinical use for leukemia induction
17
and consolidation,68,69 these results are of particular relevance clinically.27,29
18
Our results support a previous in vivo study with C1498, in which mice treated
19
with a GM-CSF-secreting irradiated cell vaccine five or seven days after
20
cytarabine treatment were protected against leukemia development, despite
21
transient development of severe neutophenia and lymphopenia following
22
chemotherapy. 60
23
24
We have demonstrated successful combination of immunotherapy following
25
chemotherapy pretreatment of leukemic mice, and have shown that
23
From www.bloodjournal.org by guest on February 4, 2015. For personal use only.
GIBBINS et al
IMMUNOTHERAPY
CHEMOTHERAPY ENABLES POTENT LEUKEMIA
1
cytarabine alone did not induce changes to the immune environment that
2
could be detected by day 20. Given the suppressive effects of C1498, we
3
expected that cytarabine pretreatment would have alleviated some of the
4
leukemia-induced suppression and consequently, more robust immune
5
responses would be detected in mice that received the combined treatment.
6
In contrast to this, we observed little improvement in vaccine-mediated
7
responses following cytarabine. We expect that although the leukemic burden
8
was certainly reduced in cytarabine treated mice, the number of circulating
9
tumor cells may have been substantially increased by time of vaccination,
10
which was delayed until nearly three weeks following chemotherapy.
11
Evidence for this is reported in a study by Lin et al., where aggressive C1498
12
progression was demonstrated by in vivo imaging, and a ten-thousand fold
13
increase in whole body photon counts of leukemic cells was observed within 2
14
weeks of tumor challenge 60. Comparable with our findings, relapse also
15
occurred in mice treated with the same dose of cytarabine we used in this
16
study, despite using half the dose of viable C1498 cells for leukemic challenge.
17
It may be possible that earlier administration of the vaccine following
18
cytarabine treatment could generate more robust immune responses in this
19
group. Since we also found little evidence to support a role for cytarabine in
20
modulating proportions of Tregs and MDSCs, the efficacy against rechallenge
21
we observe when cytarabine is combined with vaccination may simply reflect
22
the capacity of this drug to drastically reduce the burden of leukemia cells,
23
and the pre-existing immunity afforded by the vaccine before rechallenge.
24
24
From www.bloodjournal.org by guest on February 4, 2015. For personal use only.
GIBBINS et al
IMMUNOTHERAPY
CHEMOTHERAPY ENABLES POTENT LEUKEMIA
1
In comparison to vaccination with defined antigens, an α-GalCer-loaded
2
autologous whole cell vaccine for acute leukemia has the advantages of
3
stimulating both innate and adaptive immunity, and covering a broad range of
4
leukemia-associated antigens without being limited by HLA phenotype.71,72
5
Since leukemic blasts are readily obtained from the blood or bone marrow of
6
patients at diagnosis,73 and α-GalCer has been used safely in early-phase
7
clinical trials,75-77 vaccination with α-GalCer-pulsed irradiated leukemia cells
8
may represent a feasible post-remission immunotherapy. We are aware that
9
other approaches to adjuvanting autologous tumor cell vaccines have been
10
described for hematological malignancies, including toll-like receptor (TLR)
11
ligands.78 While we have not directly compared α-GalCer and TLR ligands in
12
this model, the two approaches are not mutually exclusive and it is possible
13
that these adjuvants may synergize to provide enhanced therapy 71,72.
14
15
In summary, an α-GalCer-adjuvanted whole leukemia cell vaccine that is
16
effective at preventing leukemia development in naïve animals is ineffective in
17
the presence of established leukemia, due to suppressive activities of Tregs,
18
MDSCs and the leukemia cells themselves. However, in the setting of
19
remission after cytarabine treatment, the vaccination leads to durable
20
protection against subsequent leukemia rechallenge. We suggest that post-
21
induction immunotherapy with an autologous irradiated leukemia cell vaccine
22
adjuvanted with α-GalCer may prove a useful strategy for prevention of high-
23
risk acute leukemias.
24
25
25
From www.bloodjournal.org by guest on February 4, 2015. For personal use only.
GIBBINS et al
IMMUNOTHERAPY
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
CHEMOTHERAPY ENABLES POTENT LEUKEMIA
Acknowledgements
This research is supported by the Marsden Fund Council from Government
funding, administered by the Royal Society of New Zealand, and by the New
Zealand Ministry of Science and Innovation (C08X0808).
We acknowledge the NIH Tetramer Core Facility (contract
HHSN272201300006C) for provision of CD1d tetramers and thank Chingwen
Tang and Taryn Osmond for their technical assistance.
Contribution: J.D.G., L.R.A., R.W., T.R.P. and I.F.H. designed the research;
J.D.G., L.R.A., R.W., and I.F.H. analyzed and interpreted the data and wrote
the manuscript; J.D.G. and L.R.A. conducted the experiments; G.F.P. and
B.J.C. synthesized α-GalCer.
Conflict-of-interest disclosure: The authors declare no competing financial
interests.
26
From www.bloodjournal.org by guest on February 4, 2015. For personal use only.
GIBBINS et al
IMMUNOTHERAPY
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
CHEMOTHERAPY ENABLES POTENT LEUKEMIA
References
1.
Magenau J, Couriel DR. Hematopoietic stem cell transplantation for
acute myeloid leukemia: to whom, when, and how. Curr Oncol Rep 2013;
15(5): 436-44.
2.
Terwijn M, van Putten WLJ, Kelder A, et al. High prognostic impact of
flow cytometric minimal residual disease detection in acute myeloid leukemia:
data from the HOVON/SAKK AML 42A study. J Clin Oncol 2013; 31(31):
3889-97.
3.
Grimwade D, Hills RK, Moorman AV, et al. Refinement of cytogenetic
classification in acute myeloid leukemia: determination of prognostic
significance of rare recurring chromosomal abnormalities among 5876
younger adult patients treated in the United Kingdom Medical Research
Council trials. Blood 2010; 116(3): 354-65.
4.
Schlenk RF, Döhner K, Krauter J, et al. Mutations and treatment
outcome in cytogenetically normal acute myeloid leukemia. N Engl J Med
2008; 358(18): 1909-18.
5.
Eckert C, Henze G, Seeger K, et al. Use of allogeneic hematopoietic
stem-cell transplantation based on minimal residual disease response
improves outcomes for children with relapsed acute lymphoblastic leukemia in
the intermediate-risk group. J Clin Oncol 2013; 31(21): 2736-42.
6.
Potter VT, Krishnamurthy P, Barber LD, et al. Long-Term Outcomes of
Alemtuzumab-Based Reduced-Intensity Conditioned Hematopoietic Stem Cell
Transplantation for Myelodysplastic Syndrome and Acute Myelogenous
Leukemia Secondary to Myelodysplastic Syndrome. Biol Blood Marrow
Transplant 2013.
27
From www.bloodjournal.org by guest on February 4, 2015. For personal use only.
GIBBINS et al
IMMUNOTHERAPY
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
CHEMOTHERAPY ENABLES POTENT LEUKEMIA
7.
Khera N, Zeliadt SB, Lee SJ. Economics of hematopoietic cell
transplantation. Blood 2012; 120(8): 1545-51.
8.
Switzer GE, Bruce JG, Myaskovsky L, et al. Race and ethnicity in
decisions about unrelated hematopoietic stem cell donation. Blood 2013;
121(8): 1469-76.
9.
Martner A, Thorén FB, Aurelius J, Hellstrand K. Immunotherapeutic
strategies for relapse control in acute myeloid leukemia. Blood Rev 2013;
27(5): 209-16.
10.
Walter RB, Buckley SA, Pagel JM, et al. Significance of minimal
residual disease before myeloablative allogeneic hematopoietic cell
transplantation for AML in first and second complete remission. Blood 2013;
122(10): 1813-21.
11.
Yin JAL, O'Brien MA, Hills RK, Daly SB, Wheatley K, Burnett AK.
Minimal residual disease monitoring by quantitative RT-PCR in core binding
factor AML allows risk stratification and predicts relapse: results of the United
Kingdom MRC AML-15 trial. Blood 2012; 120(14): 2826-35.
12.
Inaba H, Coustan-Smith E, Cao X, et al. Comparative analysis of
different approaches to measure treatment response in acute myeloid
leukemia. J Clin Oncol 2012; 30(29): 3625-32.
13.
Attarbaschi A, Mann G, Panzer-Grümayer R, et al. Minimal residual
disease values discriminate between low and high relapse risk in children with
B-cell precursor acute lymphoblastic leukemia and an intrachromosomal
amplification of chromosome 21: the Austrian and German acute
lymphoblastic leukemia Berlin-Frankfurt-Munster (ALL-BFM) trials. J Clin
Oncol 2008; 26(18): 3046-50.
14.
Bassan R, Spinelli O, Oldani E, et al. Improved risk classification for
risk-specific therapy based on the molecular study of minimal residual disease
(MRD) in adult acute lymphoblastic leukemia (ALL). Blood 2009; 113(18):
4153-62.
15.
Anguille S, Lion E, Smits E, Berneman ZN, Van Tendeloo VFI.
Dendritic cell vaccine therapy for acute myeloid leukemia: Questions and
answers. vaccines 2011; 7(5): 579-84.
16.
Network CGAR. Genomic and epigenomic landscapes of adult de novo
acute myeloid leukemia. N Engl J Med 2013; 368(22): 2059-74.
17.
Greiner J, Ono Y, Hofmann S, et al. Mutated regions of nucleophosmin
1 elicit both CD4(+) and CD8(+) T-cell responses in patients with acute
myeloid leukemia. Blood 2012; 120(6): 1282-9.
18.
Ochsenreither S, Fusi A, Geikowski A, et al. Wilms' tumor protein 1
(WT1) peptide vaccination in AML patients: predominant TCR CDR3
sequence associated with remission in one patient is detectable in other
vaccinated patients. Cancer Immunol Immunother 2012; 61(3): 313-22.
19.
Van Tendeloo VF, Van de Velde A, Van Driessche A, et al. Induction of
complete and molecular remissions in acute myeloid leukemia by Wilms'
tumor 1 antigen-targeted dendritic cell vaccination. Proc Natl Acad Sci USA
2010; 107(31): 13824-9.
20.
Smits ELJ, Lee C, Hardwick N, et al. Clinical evaluation of cellular
immunotherapy in acute myeloid leukaemia. Cancer Immunol Immunother
2011; 60(6): 757-69.
21.
Qiu C-H, Miyake Y, Kaise H, Kitamura H, Ohara O, Tanaka M. Novel
subset of CD8{alpha}+ dendritic cells localized in the marginal zone is
β
28
From www.bloodjournal.org by guest on February 4, 2015. For personal use only.
GIBBINS et al
IMMUNOTHERAPY
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
CHEMOTHERAPY ENABLES POTENT LEUKEMIA
responsible for tolerance to cell-associated antigens. J Immunol 2009; 182(7):
4127-36.
22.
Kuball J, de Boer K, Wagner E, et al. Pitfalls of vaccinations with WT1-,
Proteinase3- and MUC1-derived peptides in combination with
MontanideISA51 and CpG7909. Cancer Immunol Immunother 2011; 60(2):
161-71.
23.
Mattarollo SR, West AC, Steegh K, et al. NKT cell adjuvant-based
tumor vaccine for treatment of myc oncogene-driven mouse B-cell lymphoma.
Blood 2012; 120(15): 3019-29.
24.
Fujii S-I, Shimizu K, Kronenberg M, Steinman RM. Prolonged IFN- –
producing NKT response induced with -galactosylceramide–loaded DCs.
Nat Immunol 2002; 3(9): 867-74.
25.
Hunn MK, Farrand KJ, Broadley KWR, et al. Vaccination with irradiated
tumor cells pulsed with an adjuvant that stimulates NKT cells is an effective
treatment for glioma. Clinical cancer research : an official journal of the
American Association for Cancer Research 2012; 18(23): 6446-59.
26.
Weinkove R, Brooks CR, Carter JM, Hermans IF, Ronchese F.
Functional invariant natural killer T-cell and CD1d axis in chronic lymphocytic
leukemia: implications for immunotherapy. Haematologica 2013; 98(3): 37684.
27.
Chung Y, Qin H, Kang C-Y, Kim S, Kwak LW, Dong C. An NKTmediated autologous vaccine generates CD4 T-cell dependent potent
antilymphoma immunity. Blood 2007; 110(6): 2013-9.
28.
Shimizu K, Goto A, Fukui M, Taniguchi M, Fujii S-I. Tumor cells loaded
with alpha-galactosylceramide induce innate NKT and NK cell-dependent
resistance to tumor implantation in mice. J Immunol 2007; 178(5): 2853-61.
29.
Shimizu K, Kurosawa Y, Taniguchi M, Steinman RM, Fujii S-I. Crosspresentation of glycolipid from tumor cells loaded with alphagalactosylceramide leads to potent and long-lived T cell mediated immunity
via dendritic cells. The Journal of experimental medicine 2007; 204(11): 264153.
30.
Lindau D, Gielen P, Kroesen M, Wesseling P, Adema GJ. The
immunosuppressive tumour network: myeloid-derived suppressor cells,
regulatory T cells and natural killer T cells. Immunology 2013; 138(2): 105-15.
31.
Ustun C, Miller JS, Munn DH, Weisdorf DJ, Blazar BR. Regulatory T
cells in acute myelogenous leukemia: is it time for immunomodulation? Blood
2011.
32.
Barrett AJ, Le Blanc K. Immunotherapy prospects for acute myeloid
leukaemia. Clinical & Experimental Immunology 2010: no-no.
33.
Mohty M. Circulating blood dendritic cells from myeloid leukemia
patients display quantitative and cytogenetic abnormalities as well as
functional impairment. Blood 2001; 98(13): 3750-6.
34.
Curti A, Pandolfi S, Valzasina B, et al. Modulation of tryptophan
catabolism by human leukemic cells results in the conversion of CD25- into
CD25+ T regulatory cells. Blood 2007; 109(7): 2871-7.
35.
Szczepanski MJ, Szajnik M, Czystowska M, et al. Increased frequency
and suppression by regulatory T cells in patients with acute myelogenous
leukemia. Clin Cancer Res 2009; 15(10): 3325-32.
36.
Shenghui Z, Yixiang H, Jianbo W, et al. Elevated frequencies of CD4
CD25 CD127lo regulatory T cells is associated to poor prognosis in patients
α
γ
29
From www.bloodjournal.org by guest on February 4, 2015. For personal use only.
GIBBINS et al
IMMUNOTHERAPY
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
CHEMOTHERAPY ENABLES POTENT LEUKEMIA
with acute myeloid leukemia. International journal of cancer Journal
international du cancer 2011; 129(6): 1373-81.
37.
Zhou Q, Bucher C, Munger ME, et al. Depletion of endogenous tumorassociated regulatory T cells improves the efficacy of adoptive cytotoxic T-cell
immunotherapy in murine acute myeloid leukemia. Blood 2009; 114(18):
3793-802.
38.
Kissenpfennig A, Henri S, Dubois B, et al. Dynamics and function of
Langerhans cells in vivo: dermal dendritic cells colonize lymph node areas
distinct from slower migrating Langerhans cells. Immunity 2005; 22(5): 643-54.
39.
Fontenot JD, Rasmussen JP, Williams LM, Dooley JL, Farr AG,
Rudensky AY. Regulatory T cell lineage specification by the forkhead
transcription factor foxp3. Immunity 2005; 22(3): 329-41.
40.
Bradner WT, Pindell MH. Myeloid Leukemia C1498 as a Screen for
Cancer Chemotherapeutic Agents. 1966; (26): 375-90.
41.
Plettenburg O, Bodmer-Narkevitch V, Wong C-H. Synthesis of alphagalactosyl ceramide, a potent immunostimulatory agent. J Org Chem 2002;
67(13): 4559-64.
42.
Giaccone G, Punt CJA, Ando Y, et al. A phase I study of the natural
killer T-cell ligand alpha-galactosylceramide (KRN7000) in patients with solid
tumors. Clinical cancer research : an official journal of the American
Association for Cancer Research 2002; 8(12): 3702-9.
43.
Prendergast KA, Osmond TL, Ochiai S, Hermans IF, Kirman JR.
Sustained in vivo depletion of splenic langerin(+) CD8 (+) dendritic cells is
well-tolerated by lang-DTREGFP mice. J Immunol Methods 2014.
44.
Farrand KJ, Dickgreber N, Stoitzner P, Ronchese F, Petersen TR,
Hermans IF. Langerin(+)CD8 alpha(+) Dendritic Cells Are Critical for CrossPriming and IL-12 Production in Response to Systemic Antigens. J Immunol
2009; 183(12): 7732-42.
45.
Idoyaga J, Suda N, Suda K, Park CG, Steinman RM. Antibody to
Langerin/CD207 localizes large numbers of CD8 alpha(+) dendritic cells to the
marginal zone of mouse spleen. P Natl Acad Sci Usa 2009; 106(5): 1524-9.
46.
Petersen TR, Sika-Paotonu D, Knight DA, Simkins HMA, Hermans IF.
Exploiting the role of endogenous lymphoid-resident dendritic cells in the
priming of NKT cells and CD8+ T cells to dendritic cell-based vaccines. Plos
One 2011; 6(3): e17657.
47.
Hermans IF, Silk JD, Gileadi U, et al. NKT cells enhance CD4+ and
CD8+ T cell responses to soluble antigen in vivo through direct interaction
with dendritic cells. J Immunol 2003; 171(10): 5140-7.
48.
Silk JD, Hermans IF, Gileadi U, et al. Utilizing the adjuvant properties
of CD1d-dependent NK T cells in T cell-mediated immunotherapy. J Clin
Invest 2004; 114(12): 1800-11.
49.
Fujii S-I, Liu K, Smith C, Bonito AJ, Steinman RM. The linkage of
innate to adaptive immunity via maturing dendritic cells in vivo requires CD40
ligation in addition to antigen presentation and CD80/86 costimulation. The
Journal of experimental medicine 2004; 199(12): 1607-18.
50.
Liu K, Idoyaga J, Charalambous A, et al. Innate NKT lymphocytes
confer superior adaptive immunity via tumor-capturing dendritic cells. The
Journal of experimental medicine 2005; 202(11): 1507-16.
α
30
From www.bloodjournal.org by guest on February 4, 2015. For personal use only.
GIBBINS et al
IMMUNOTHERAPY
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
CHEMOTHERAPY ENABLES POTENT LEUKEMIA
51.
Simkins HMA, Hyde E, Farrand KJ, et al. Administration of alphagalactosylceramide impairs the survival of dendritic cell subpopulations in vivo.
Journal of Leukocyte Biology 2011; 89(5): 753-62.
52.
Fallarino F, Grohmann U, You S, et al. The combined effects of
tryptophan starvation and tryptophan catabolites down-regulate T cell receptor
zeta-chain and induce a regulatory phenotype in naive T cells. J Immunol
2006; 176(11): 6752-61.
53.
Curti A, Pandolfi S, Valzasina B, et al. Modulation of tryptophan
catabolism by human leukemic cells results in the conversion of CD25- into
CD25+ T regulatory cells. Blood 2006: 1-8.
54.
Nagaraj S, Schrum AG, Cho H-I, Celis E, Gabrilovich DI. Mechanism of
T cell tolerance induced by myeloid-derived suppressor cells. J Immunol
2010; 184(6): 3106-16.
55.
Christiansson L, Söderlund S, Svensson E, et al. Increased level of
myeloid-derived suppressor cells, programmed death receptor ligand
1/programmed death receptor 1, and soluble CD25 in Sokal high risk chronic
myeloid leukemia. PLoS ONE 2013; 8(1): e55818.
56.
Mattarollo SR, Steegh K, Li M, Duret H, Foong Ngiow S, Smyth MJ.
Transient Foxp3(+) regulatory T-cell depletion enhances therapeutic
anticancer vaccination targeting the immune-stimulatory properties of NKT
cells. Immunol Cell Biol 2013; 91(1): 105-14.
57.
Bhattacharya K, Chandra S, Mandal C. Critical Stoichiometric ratio of
CD4(+) CD25(+) FoxP3(+) Treg and CD4(+) CD25(-) Tresp persuades
immunosuppression in patients with B-cell Acute Lymphoblastic Leukemia.
Immunology 2013.
58.
Setiady YY, Coccia JA, Park PU. In vivo depletion of CD4+FOXP3+
Treg cells by the PC61 anti-CD25 monoclonal antibody is mediated by
FcgammaRIII+ phagocytes. Eur J Immunol 2010; 40(3): 780-6.
59.
Kanakry CG, Hess AD, Gocke CD, et al. Early lymphocyte recovery
after intensive timed sequential chemotherapy for acute myelogenous
leukemia: peripheral oligoclonal expansion of regulatory T cells. Blood 2011;
117(2): 608-17.
60.
Lin JM, Li B, Rimmer E, VanRoey M, Jooss K. Enhancement of the
anti-tumor efficacy of a GM-CSF-secreting tumor cell immunotherapy in
preclinical models by cytosine arabinoside. Exp Hematol 2008; 36(3): 319-28.
61.
Phillips GL, Reece DE, Shepherd JD, et al. High-dose cytarabine and
daunorubicin induction and postremission chemotherapy for the treatment of
acute myelogenous leukemia in adults. Blood 1991; 77(7): 1429-35.
62.
Bishop JF, Matthews JP, Young GA, et al. A randomized study of highdose cytarabine in induction in acute myeloid leukemia. Blood 1996; 87(5):
1710-7.
63.
Boyer MW, Orchard PJ, Gorden KB, Anderson PM, Mclvor RS, Blazar
BR. Dependency on intercellular adhesion molecule recognition and local
interleukin-2 provision in generation of an in vivo CD8+ T-cell immune
response to murine myeloid leukemia. Blood 1995; 85(9): 2498-506.
64.
Fujii S-I, Shimizu K, Smith C, Bonifaz L, Steinman RM. Activation of
natural killer T cells by alpha-galactosylceramide rapidly induces the full
maturation of dendritic cells in vivo and thereby acts as an adjuvant for
combined CD4 and CD8 T cell immunity to a coadministered protein. The
Journal of experimental medicine 2003; 198(2): 267-79.
31
From www.bloodjournal.org by guest on February 4, 2015. For personal use only.
GIBBINS et al
IMMUNOTHERAPY
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
CHEMOTHERAPY ENABLES POTENT LEUKEMIA
65.
Mussai F, De Santo C, Abu-Dayyeh I, et al. Acute myeloid leukemia
creates an arginase-dependent immunosuppressive microenvironment. Blood
2013; 122(5): 749-58.
66.
Curti A, Trabanelli S, Onofri C, et al. Indoleamine 2,3-dioxygenaseexpressing leukemic dendritic cells impair a leukemia-specific immune
response by inducing potent T regulatory cells. Haematologica 2010; 95(12):
2022-30.
67.
Fu J, Zhang Z, Zhou L, et al. Impairment of CD4+ cytotoxic T cells
predicts poor survival and high recurrence rates in patients with hepatocellular
carcinoma. Hepatology 2013; 58(1): 139-49.
68.
Rowe JM, Tallman MS. How I treat acute myeloid leukemia. Blood
2010; 116(17): 3147-56.
69.
Frcpath PAV, Phd NG, Msc RW, et al. Treatment reduction for children
and young adults with low-risk acute lymphoblastic leukaemia defined by
minimal residual disease (UKALL 2003): a randomised controlled trial. Lancet
Oncology 2013; 14(3): 199-209.
70.
Markasz L, Skribek H, Uhlin M, et al. Effect of frequently used
chemotherapeutic drugs on cytotoxic activity of human cytotoxic Tlymphocytes. J Immunother 2008; 31(3): 283-93.
71.
Ando T, Ito H, Ohtaki H, Seishima M. Toll-like receptor agonists and
alpha-galactosylceramide synergistically enhance the production of interferongamma in murine splenocytes. Scientific reports 2013; 3: 2559.
72.
Hermans IF, Silk JD, Gileadi U, et al. Dendritic cell function can be
modulated through cooperative actions of TLR ligands and invariant NKT cells.
J Immunol 2007; 178(5): 2721-9.
73.
Cripe LD. Adult acute leukemia. Curr Probl Cancer 1997; 21(1): 1-64.
74.
Metelitsa LS, Weinberg KI, Emanuel PD, Seeger RC. Expression of
CD1d by myelomonocytic leukemias provides a target for cytotoxic NKT cells.
Leukemia 2003; 17(6): 1068-77.
75.
Nieda M, Okai M, Tazbirkova A, et al. Therapeutic activation of
Valpha24+Vbeta11+ NKT cells in human subjects results in highly
coordinated secondary activation of acquired and innate immunity. Blood
2004; 103(2): 383-9.
76.
Ishikawa A, Motohashi S, Ishikawa E, et al. A phase I study of alphagalactosylceramide (KRN7000)-pulsed dendritic cells in patients with
advanced and recurrent non-small cell lung cancer. Clinical cancer research :
an official journal of the American Association for Cancer Research 2005;
11(5): 1910-7.
77.
Richter J, Neparidze N, Zhang L, et al. Clinical regressions and broad
immune activation following combination therapy targeting human NKT cells
in myeloma. Blood 2013; 121(3): 423-30.
78.
Goldstein MJ, Varghese B, Brody JD, et al. A CpG-loaded tumor cell
vaccine induces antitumor CD4+ T cells that are effective in adoptive therapy
for large and established tumors. Blood 2011; 117(1): 118-27.
Figure Legends
32
From www.bloodjournal.org by guest on February 4, 2015. For personal use only.
GIBBINS et al
IMMUNOTHERAPY
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
CHEMOTHERAPY ENABLES POTENT LEUKEMIA
Figure 1) An α-GalCer-pulsed whole leukemia cell vaccine protects against
acute leukemia and is dependent on CD4+ and CD8+ T cells and langerin+
CD8α+ DCs. A vaccine comprising α-GalCer-pulsed irradiated C1498 cells was
administered intravenously seven days before C1498 challenge. (A) Bone marrow
histology, (B) peripheral blood smear and (C) peripheral white blood cell counts were
performed at symptom onset in unvaccinated animals, 40 days after C1498
challenge in vaccinated animals, and in naïve control mice. (D) Kaplan Meier plot
showing symptom-free survival of vaccinated and unvaccinated mice after leukemia
administration on day zero. Symbols represent treatments: unvaccinated ( ),
vaccinated with α-GalCer-pulsed irradiated leukemia cells ( ), vaccinated with
unpulsed irradiated leukemia cells ( ), vaccinated with free α-GalCer ( ). Statistical
analysis compares unvaccinated and vaccinated with α-GalCer-pulsed irradiated
leukemia cells groups. (E) Kaplan Meier plots showing symptom-free survival of mice
vaccinated with leukemia/α-GalCer and challenged with C1498 at day zero. Symbols
represent treatments: unvaccinated ( ), vaccinated with α-GalCer-pulsed irradiated
leukemia cells ( ), depletion of CD4+ cells ( ) or depletion of CD8+ cells ().
Statistical analyses compares the depletion groups to mice vaccinated with αGalCer-pulsed irradiated leukemia cells. (F) Lang-EGFPDTR mice were
prophylactically vaccinated and one group was administered DT. Symbols represent
treatment groups: unvaccinated ( ), prophylactic α-GalCer vaccination ( ),
prophylactic vaccination and DT treatment (). Symptom-free survival was analyzed
and is graphed. *P<0.05 (one-way ANOVA with a Bonferroni post test). Figure A-C
represents a single experiment, Figure D represents three experiments and Figures
E-F represent two experiments; five mice per group were used for each experiment.
**P<.01 (Mantel-Cox log-rank test). *P<.05, ***P<.001 (Mantel-Cox log-rank test).
Figure 2) Leukemia/α-GalCer vaccination is ineffective in the presence of
established acute leukemia despite NKT cell and DC activation. Mice were
challenged with C1498 cells i.v. one week before vaccination with α-GalCer-pulsed
or unpulsed irradiated leukemia cells. (A) Kaplan Meier graph showing survival of
mice. Symbols represent treatment groups: unvaccinated ( ), therapeutic α-GalCer
vaccination ( ), treatment with unpulsed irradiated leukemia cells ( ). This graph
represents three experiments, each with five mice per group. (B-E) Mice were
inoculated with C1498 cells seven or fourteen days before vaccination with irradiated
α-GalCer-pulsed leukemia cells. (B) Representative flow cytometry plots showing
33
From www.bloodjournal.org by guest on February 4, 2015. For personal use only.
GIBBINS et al
IMMUNOTHERAPY
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
CHEMOTHERAPY ENABLES POTENT LEUKEMIA
identification of splenic NKT cells (CD3+ α-GalCer–loaded CD1d tetramer+). (C)
Frequency of splenic NKT cells following vaccination in mice with and without
established acute leukemia. (D) Serum IL-4 levels and (E) serum IFN-γ levels two
hours after vaccination (F-G) The splenic langerin+ CD8α+ DC population in LangEGFP mice was analyzed 24 hours following vaccination. (F) Representative flow
cytometry plots showing identification of splenic langerin+ CD8α+ DCs (CD11c+
GFP+). (G) Frequency of splenic langerin+ CD8α+ DCs. (H-I) The expression of CD40
and CD86 on langerin+ CD8α+ DCs respectively. (J-K) The CD8α+ DC population in
C57BL/6 mice was analyzed 24 hours following vaccination. The expression of CD40
and CD86 on CD8α+ DCs, respectively. (L) Serum IL-12p70 was quantified five hours
following vaccination. These results are indicative of two independent experiments,
each with five mice per group. **P<.01, ***P<.001 (one-way ANOVA with a
Bonferroni post test).
Figure 3) Established leukemia disrupts leukemia/α-GalCer vaccine-mediated
CD4+ T cell function. Mice were challenged with C1498 i.v. seven days before
vaccination and responses were analyzed one week later. (A) Splenocytes were
cultured for 24 hours with () or without () DCs loaded with C1498 lysate.
Supernatant IFN-γ was quantified. (B-F) The splenic CD4+ and CD8+ T cell
populations were analyzed by flow cytometry. (B) Representative flow cytometry
plots showing identification of CD4+ and CD8+ T cells from mouse spleens. IFN-γ+
cells were determined by comparison to an isotype control antibody (lower left). (C,
E) MFI of CD44 expressed on CD8+ and CD4+ T cells respectively. (D, F) The
proportion of CD8+ and CD4+ cells producing IFN-γ respectively. Panel A represents
three experiments. Statistical analysis compares experiments performed in the
presence of DCs only (one-way ANOVA, Bonferroni post test) and panels B-F
represent two experiments, each with five mice per group. *P<.05, **P<.01,
***P<.001 (one-way ANOVA with a Bonferroni post test).
Figure 4) Elevated immune suppression in hosts with established acute
leukemia. (A-B) Mice were challenged with C1498 i.v. and the immune response in
the liver and spleen was analyzed 20 days later. (A) Flow cytometric identification of
Tregs. (B) Percentage of Tregs in the spleens and livers of naïve and leukemiachallenged mice. (C) Symptom-free survival of mice challenged C1498 and
vaccinated seven days later. One group was depleted of Tregs the day after
34
From www.bloodjournal.org by guest on February 4, 2015. For personal use only.
GIBBINS et al
IMMUNOTHERAPY
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
CHEMOTHERAPY ENABLES POTENT LEUKEMIA
leukemia challenge. Symbols represent treatment groups: unvaccinated ( ),
therapeutic α-GalCer vaccination ( ), therapeutic α-GalCer vaccination plus PC61
( ), irradiated C1498 cells plus PC61 (). Statistical analysis compares therapeutic
α-GalCer vaccination plus PC61 to therapeutic α-GalCer vaccination. (D-G) mice
were challenged C1498 i.v. and euthanized 20 days later. (D) Flow cytometric
identification of splenic CD11b+ Ly6G+ MDSCs. (E) The proportion of MDSCs within
live splenocytes. (F,G) Splenic CD11b+ cells were isolated from naïve or C1498
challenged mice and cultured with CFSE labeled lymphocytes and anti-CD3 and antiCD28. A representative histogram of CFSE dilution of CD4+ cells (F) and CD8+ cells
(G) incubated with CD11b cells from naïve mice (black line) or C1498 challenged
mice (dotted line); unstimulated (shaded) and graph of reduced CFSE. (H-I) CFSE
labeled lymph node cells stimulated with anti-CD3 and anti-CD28 were cultured with
naïve splenocytes or C1498 cells for 72 hours. A representative histogram of CFSE
dilution of CD4+ (H) and CD8+ (I) T cells culted with naïve splenocytes (black line) or
C1498 cells (dotted line); unstimulated (shaded). The percent divided of CD4+ cells
(H) or CD8+ cells (I). This figure represents three experiments, each with five mice
per group. Panels B, and E-H *P<.05, **P<.01 (t-test with Mann Whitney). Panel C
*P<.05 (log-rank Mantel-Cox test).
Figure 5) Chemotherapy restores the T cell compartment in leukemic mice.
Mice were challenged with C1498 cells and 24 hours three 3 mg doses of cytarabine
were administered i.p. ten hours apart. (A-D) The T cell populations in the spleen
were analyzed and identified as CD3+ expressing either CD8 or CD4. The proportion
of live cells expressing CD3 and CD8 (A) or CD3 and CD4 (B). The MFI of CD44
CD8+ cells (C) and CD4+ cells (D). A-D represents three experiments, with five mice
per group *P<.05, **P<.01 (one-way ANOVA with a Bonferroni post test).
Figure 6) Cytarabine pretreatment does not suppress vaccine-induced immune
responses. Mice were challenged with C1498 cells and 24 hours three 3 mg doses
of cytarabine were administered i.p. ten hours apart. On day 23 one group of
chemotherapy treated mice was vaccinated with leukemia/α-GalCer and responses
were analyzed one week later. The percentage of CD4+ FoxP3+ cells of CD3+ in the
spleen (A) and liver (B). The percentage of CD11b+ Ly6G+ of CD3- in the spleen (C)
and liver (D). The ratio of CD44hi CD8+ effector T cells to CD4+ FoxP3+ Tregs and
ratio of CD44hi CD4+ effector T cells to CD4+ FoxP3+ Tregs in the spleen (E, G) and
liver (F,H), respectively. (I) The percentage of CD3+ T cells in the bone marrow. The
35
From www.bloodjournal.org by guest on February 4, 2015. For personal use only.
GIBBINS et al
IMMUNOTHERAPY
1
2
3
4
5
6
7
8
9
10
11
12
13
14
CHEMOTHERAPY ENABLES POTENT LEUKEMIA
percentage of PD-1+ CD8+ and CD4+ T cells in the bone marrow (J, K), respectively.
Graphs represent one experiment, with five-six mice per group *P<.05, **P<.01,
***P<.001 (one-way ANOVA with a Bonferroni post test).
Figure 7) Vaccination following chemotherapy protects against leukemic
rechallenge. (A-B) Mice were challenged with C1498 cells and 24 hours three 3 mg
doses of cytarabine were administered i.p. ten hours apart. On day 23 one group of
chemotherapy treated mice were administered the leukemia/α-GalCer vaccine and
symptom free survival was monitored. (A,C) Surviving mice were rechallenged on
day 45 with a five-fold increased dose of 5 x 105 viable C1498 cells and symptomfree survival was again followed. Symbols represent treatment groups: leukemia only
(), n = 10, cytarabine treatment ( ) n =19, cytarabine plus α-GalCer vaccination ()
n = 20. B-C represent two experiments *P<.05, **P<.01 (one-way ANOVA with a
Bonferroni post test). P<.0001 (Log-rank Mantel-Cox Test).
15
36
Figure 1
A
Naive
C1498 alone
Vaccine + C1498
25 μ
μM
Naive
C1498 alone
Vaccine + C1498
E
*
60
*
40
20
0
C1498
Vaccine
-
+
-
80
60
40
*
20
*
50
Time (days)
**
80
60
40
20
0
0
10
100
20
30
40
50
40
50
Time (days)
F
0
100
+
+
100
0
Symptom Free Survival (%)
D
80
Symptom Free Survival (%)
C
White blood cells per litre
of blood (x109)
50 μ
μM
Symptom Free Survival (%)
B
100
***
80
60
40
20
0
0
10
20
30
Time (days)
Figure 2
80
85.2
CD1d tet
FSC-A
20
40
60
80
1000
500
1500
200
+
I
***
+
+
+
***
4000
L
***
2000
500
1000
0
C1498 14 days +
Vaccine
+
+
+
K
J
3000
***
***
**
2000
0
C1498 7 days C1498 14 days +
Vaccine
+
+
+
4000
1000
+
+
+
+
+
0
C1498 7 days C1498 14 days +
Vaccine
4000
**
***
**
3000
2000
0
C1498 7 days C1498 14 days +
Vaccine
0
6000
2000
5000
1000
C1498 14 days +
Vaccine
-
+
+
+
15
+
+
+
+
+
+
+
**
10
5
C1498 14 days +
Vaccine
+
+
3000
1000
0.354
Langerin
FSC-A
C1498 7 days C1498 14 days +
Vaccine
+
+
+
+
58.3
IL-12 pg/ml
CD40 MFI on Lang+ CD11c+
Cells
C1498 7 days C1498 14 days +
Vaccine
400
+
+
+
+
600
0
0
+
G
SSC-A
1500
0
F
***
800
IFN-y (pg/ml)
**
E
***
CD86 MFI on Lang+ CD11c+
Cells
2000
**
5
C1498 7 days
C1498 14 days +
Vaccine
100
MFI of CD86 on CD8+ CD11c+
Cells
IL-4 pg/ml
2500
10
CD11c
0
D
CD40 MFI on CD8+ CD11c+
Cells
**
Percent of CD11c+ Cells
Expressing Langerin
20
CD3
2.53
40
0
***
20
From www.bloodjournal.org by guest on February 4, 2015. For15personal use only.
60
Time (days)
H
Percent of CD3+ cells
that are tetramer positive
C
B
100
SSC-A
Symptom Free Survival (%)
A
+
+
+
Figure 3
C
600
400
400
200
200
0
C1498
Vaccine
E
*
800
600
***
**2015. For personal use only.
From
www.bloodjournal.org
by
guest
on
February
4,
**
MFI of CD44 on CD3+
CD4+ cells
IFN-y (pg/ml)
800
MFI of CD44 on CD3+
CD8+ cells
A
+
B
C1498
Vaccine
+
+
***
600
400
200
0
0
+
-
***
+
-
+
C1498
Vaccine
+
+
+
-
+
+
+
D
CD8
CD3
SSC-A
87.9
FSC-H
FSC-A
CD4
23.3
CD8
*
30
20
10
C1498
Vaccine
IFN-γ
IFN-γ
0.332
CD8
51
F
40
0
+
-
+
+
+
Percent of CD4+ cells that
are IFN- +
39
Percent of CD8+ cells that
are IFN- +
33.6
C1498
Vaccine
4
***
**
3
2
1
0
+
-
+
+
+
**
50
C
**
Symptom Free Survival (%)
B
Symptom Free Survival (%)
A
Percent of CD4+ cells that
are FoxP3+
Figure 4
100
FoxP3
20
10
0
Naive
C1498
Naive
SSC-A
CD11b
E
FSC-A
100
Percent divided of
CD4+ cells
% of Max
60
40
20
***
20
0
0
10
Spleen
H
**
20
30
Time (days)
1.0
0.5
60
40
20
Naive
0 101
AML
40
30
20
102
103
104
G
103
104
105
60
40
0
0
CFSE
C1498
Naive
C1498
100
Percent divided of
CD8+ cells
100
80
% of Max
Naive
60
40
20
0
0
2
10
3
10
CFSE
4
10
10
5
95
90
85
20
0
0
20
0 10
1
10
2
10
3
CFSE
10
4
10
5
40
**
40
20
Naive
C1498
*
100
80
20
40
60
0
105
100
10
**
60
80
CFSE
I
80
Time (days)
0
0.0
0
0 102
40
100
100
100
80
1.5
*
50
80
40
% of Max
F
Ly6G
2.0
60
% of Max
D
Percent of live cells that
are CD11b+ Ly6G+
Liver
C1498
80
Percent divided of
CD4+ cells
FSC-A
30
Percent divided of
CD8+ cells
CD4
SSC-A
40
From www.bloodjournal.org
by guest on February 4, 2015. For personal use only.
80
60
40
20
0
Naive
C1498
60
**
**
10
5
0
C1498
Cytarabine
-
+
-
**
2000
MFI of CD44 on CD3+
CD8+ cells
C
+
+
**
***
15
10
5
0
AML
Cytarabine
D
*
1500
-
+
-
**
3000
+
+
*
2000
1000
1000
500
0
AML
Cytarabine
Percent of live cells that are
CD3+ CD4+ cells
B
From www.bloodjournal.org by guest
20 on February 4, 2015. For personal use only.
15
MFI of CD44 on CD3+
CD4+ cells
A
Percent of live cells that are
CD3+ CD8+ cells
Figure 5
0
-
+
-
+
+
AML
Cytarabine
-
+
-
+
+
Figure 6
4
2
0
+
-
+
+
-
+
+
2
N.S.
1
0
C1498
Vaccine
Cytarabine
+
+
+
Percent of CD3+ T cells
N.S.
6
C1498
Vaccine
Cytarabine
+
-
+
+
+
+
-
+
+
+
N.S.
2
1
0
+
-
+
+
+
+
-
J
20
N.S.
15
10
5
0
+
-
+
+
-
+
+
+
+
+
10
5
-
+
-
+
+
+
-
+
+
-
H
N.S.
***
15
10
Ratio of
C1498
Vaccine
Cytarabine
5
0
-
+
-
+
+
+
-
+
+
-
+
+
+
K
**
N.S.
20
10
2
0
+
-
-
+
+
+
-
+
+
-
+
+
+
***
30
20
10
-
+
-
+
+
+
-
+
+
-
+
+
+
**
*
60
N.S.
40
20
0
-
+
-
+
+
+
-
+
+
-
***
****
200
+
+
+
N.S.
150
100
50
C1498
Vaccine
Cytarabine
N.S.
N.S.
*
40
0
C1498
Vaccine
Cytarabine
+
+
+
***
30
0
CD4+
eff/Tregs
N.S.
15
C1498
Vaccine
Cytarabine
G
***
F
Ratio of CD8+ eff/Tregs
20
Ratio of CD4+ eff/Tregs
Ratio of CD8+ eff/Tregs
E
4
C1498
Vaccine
Cytarabine
*
**
**
*
0
C1498
Vaccine
Cytarabine
+
+
+
N.S.
**
***
Percent of CD3- cells that
are CD11b+ Ly6G+
3
C1498
Vaccine
Cytarabine
*
6
C1498
Vaccine
Cytarabine
D
Percent of CD3- cells that
are CD11b+ Ly6G+
C
I use only.
B
by guest on February 4, 2015. For personal
** From www.bloodjournal.org
*
*
*
3
8
Percent PD-1+ of CD8+
T cells
8
*
N.S.
Percent PD-1+ of CD4+
T cells
Percent of CD3+ that are
FoxP3+ CD4+ T cells
A
Liver
Spleen
Percent of CD3+ that are
CD4+ FoxP3+ cells
N.S.
0
-
+
-
+
+
+
-
+
+
-
+
+
+
C1498
Vaccine
Cytarabine
-
+
-
+
+
+
-
+
+
-
+
+
+
Figure 7
A
Cytarabine
Vaccination
From www.bloodjournal.org
byC1498
guestrechallenge
on February 4, 2015. For personal use only.
Monitor
Symptom-free Survival
C
****
100
80
60
40
20
0
0
10
20
30
Time (days)
40
50
Symptom Free Survival (%)
B
Symptom Free Survival (%)
C1498 challenge
100
80
****
60
40
20
0
0
20
40
Time (days)
60
From www.bloodjournal.org by guest on February 4, 2015. For personal use only.
Prepublished online September 18, 2014;
doi:10.1182/blood-2014-04-568956
An autologous leukemia cell vaccine prevents murine acute leukemia
relapse after cytarabine treatment
John D. Gibbins, Lindsay R. Ancelet, Robert Weinkove, Benjamin J. Compton, Gavin F. Painter, Troels R.
Petersen and Ian F. Hermans
Information about reproducing this article in parts or in its entirety may be found online at:
http://www.bloodjournal.org/site/misc/rights.xhtml#repub_requests
Information about ordering reprints may be found online at:
http://www.bloodjournal.org/site/misc/rights.xhtml#reprints
Information about subscriptions and ASH membership may be found online at:
http://www.bloodjournal.org/site/subscriptions/index.xhtml
Advance online articles have been peer reviewed and accepted for publication but have not yet
appeared in the paper journal (edited, typeset versions may be posted when available prior to
final publication). Advance online articles are citable and establish publication priority; they are
indexed by PubMed from initial publication. Citations to Advance online articles must include
digital object identifier (DOIs) and date of initial publication.
Blood (print ISSN 0006-4971, online ISSN 1528-0020), is published weekly by the American Society of
Hematology, 2021 L St, NW, Suite 900, Washington DC 20036.
Copyright 2011 by The American Society of Hematology; all rights reserved.

Download Report