University of Miami
Scholarly Repository
Open Access Dissertations
Electronic Theses and Dissertations
2014-07-14
Combinatorial Therapeutic Vaccination Strategies
for Hematologic Malignancies in the Early Period
following Experimental Autologous Hematopoietic
Stem Cell Transplantation
Robert G. Newman
University of Miami, [email protected]
Follow this and additional works at: http://scholarlyrepository.miami.edu/oa_dissertations
Recommended Citation
Newman, Robert G., "Combinatorial Therapeutic Vaccination Strategies for Hematologic Malignancies in the Early Period following
Experimental Autologous Hematopoietic Stem Cell Transplantation" (2014). Open Access Dissertations. 1283.
http://scholarlyrepository.miami.edu/oa_dissertations/1283
This Open access is brought to you for free and open access by the Electronic Theses and Dissertations at Scholarly Repository. It has been accepted for
inclusion in Open Access Dissertations by an authorized administrator of Scholarly Repository. For more information, please contact
[email protected].
UNIVERSITY OF MIAMI
COMBINATORIAL THERAPEUTIC VACCINATION STRATEGIES FOR
HEMATOLOGIC MALIGNANCIES IN THE EARLY PERIOD FOLLOWING
EXPERIMENTAL AUTOLOGOUS HEMATOPOIETIC STEM CELL
TRANSPLANTATION
By
Robert G. Newman
A DISSERTATION
Submitted to the Faculty
of the University of Miami
in partial fulfillment of the requirements for
the degree of Doctor of Philosophy
Coral Gables, Florida
August 2014
©2014
Robert G. Newman
All Rights Reserved
UNIVERSITY OF MIAMI
A dissertation submitted in partial fulfillment of
the requirements for the degree of
Doctor of Philosophy
COMBINATORIAL THERAPEUTIC VACCINATION STRATEGIES FOR
HEMATOLOGIC MALIGNANCIES IN THE EARLY PERIOD FOLLOWING
EXPERIMENTAL AUTOLOGOUS HEMATOPOIETIC STEM CELL
TRANSPLANTATION
Robert G. Newman
Approved:
_____________________________
Robert B. Levy, Ph.D.
Professor of Microbiology
and Immunology
_____________________________
Krishna V. Komanduri, M.D.
Professor of Medicine
_____________________________
Eckhard R. Podack, M.D., Ph.D.
Professor of Microbiology
and Immunology
_____________________________
M. Brian Blake, Ph.D.
Dean of the Graduate School
_____________________________
Eli Gilboa, Ph.D.
Professor of Microbiology
and Immunology
NEWMAN, ROBERT G.
Combinatorial Therapeutic Vaccination Strategies for
Hematologic Malignancies in the Early Period following
Experimental Autologous Hematopoietic Stem Cell
Transplantation
(Ph.D., Cancer Biology)
(August 2014)
Abstract of a dissertation at the University of Miami.
Dissertation supervised by Professor Robert B. Levy.
No. of pages in text. (84)
Tumor relapse is the primary cause of mortality in patients with hematologic
cancers following autologous hematopoietic stem cell transplantation (HSCT).
Vaccination early post-HSCT can exploit both the state of lymphopenia and minimal
residual disease for the generation of anti-tumor immunity. Here, multiple vaccinations
utilizing lymphoma cells engineered to secrete the heat shock protein fusion gp96-Ig
within two weeks of T cell replete syngeneic HSCT led to antigen cross-presentation and
increased survival of lymphoma bearing mice. To enhance vaccine efficacy, IL-2 was
directed to predominantly memory phenotype CD8+ T lymphocytes and natural killer
cells via administration bound to anti-IL-2 monoclonal antibody clone S4B6 (IL-2S4B6).
Combination therapy with gp96-Ig vaccination and coordinated infusions of IL-2S4B6
resulted in a marked prolongation of median survival time and overall survival, which
directly correlated with a ~500% increase in effector CD8+ T cell numbers. Notably, this
dual regimen elicited large increases in both donor CD8+ T lymphocytes and natural
killer cells, but not CD4+ T lymphocytes, the former two populations essential for both
vaccine efficacy and protection against opportunistic infections post-HSCT. Indeed, IL2S4B6 treated HSCT recipients infected with Listeria monocytogenes exhibited decreased
bacterial levels. These pre-clinical studies validate a new strategy particularly well suited
to the post-HSCT environment, which may rapidly augment adaptive and innate immune
function in patients with malignant disease following autologous HSCT.
TABLE OF CONTENTS
Page
LIST OF FIGURES AND TABLES.................................................................................. iv
LIST OF PUBLICATIONS .............................................................................................. vii
CHAPTER I: Introduction .................................................................................................. 1
Section 1: Hematologic Malignancies and Current Treatment ........................................ 1
Section 2: Hematopoietic Stem Cell Transplantation for Hematologic Malignancies .... 3
Section 3: Vaccination for Hematologic Malignancies following Autologous HSCT .... 5
Section 4: Interleukin-2.................................................................................................... 7
Section 5: Overall Rationale and Hypothesis .................................................................. 9
CHAPTER II: Heat shock protein vaccination and directed IL-2 therapy amplify tumor
immunity rapidly following bone marrow transplantation in mice .................................. 12
Section 1: Summary ....................................................................................................... 12
Section 2: Background ................................................................................................... 13
Section 3: Methods ........................................................................................................ 16
Section 4: Results........................................................................................................... 19
Section 5: Discussion ..................................................................................................... 27
Section 6: Future Directions .......................................................................................... 34
FIGURES AND TABLES ................................................................................................ 36
WORKS CITED ............................................................................................................... 69
iii
LIST OF FIGURES AND TABLES
Figure A. Cellular origin of human B-cell and T-cell malignancies .................................10
Figure B. Indications for Hematopoietic Stem Cell Transplants in the US, 2011 .............11
Figure C. Transplant Activity in the US ............................................................................11
Figure 1A-B. Peritoneal vaccination with tumor cells secreting gp96-Ig in syngeneic
HSCT recipients led to the infiltration of myeloid cells and expansion of tumor-reactive
CD8+ T lymphocytes in the peritoneal cavity ....................................................................36
Figure 1C. A second vaccination with tumor cells secreting gp96-Ig led to accumulation
of tumor-reactive CD8+ T lymphocytes in secondary lymphoid organs ...........................37
Figure 1D. Equivalent expansion of tumor-reactive CD8+ T lymphocytes following
vaccination up to 2 wk post-HSCT ....................................................................................38
Figure 1E. Tumor-reactive CD8+ T lymphocytes are activated following vaccination ....39
Figure 1F. Vaccination results in functional tumor-reactive CD8+ T lymphocytes ..........40
Figure 2A-B. B7 costimulation was required for optimal tumor-reactive CD8+ T
lymphocyte expansion induced by vaccination with gp96-Ig secreting tumor cells
following syngeneic HSCT ................................................................................................41
Figure 2C. Batf3 was required for optimal tumor-reactive CD8+ T lymphocyte expansion
induced by vaccination with gp96-Ig secreting tumor cells following syngeneic HSCT..42
Figure 2D. Perforin-1 was required for optimal tumor-reactive CD8+ T lymphocyte
expansion induced by vaccination with gp96-Ig secreting tumor cells following syngeneic
HSCT .................................................................................................................................43
Figure 3A. CD4+ and CD8+ splenic and LN T cells from a typical tumor bearing donor
mouse .................................................................................................................................44
Figure 3B-C. Tumor-reactive CD8+ T lymphocytes obtained from tumor bearing donors
were expanded following transplantation into syngeneic HSCT recipients and vaccination
with tumor cells secreting gp96-Ig ....................................................................................45
Figure 3D. Tumor-reactive CD8+ T lymphocytes obtained from tumor bearing donors
were functional following transplantation into syngeneic HSCT recipients and
vaccination with tumor cells secreting gp96-Ig .................................................................46
Figure 4A. Development of minimal residual lymphoma model following syngeneic
HSCT .................................................................................................................................47
iv
Figure 4B. Multiple vaccinations with tumor cells secreting gp96-Ig expanded tumorreactive CD8+ T lymphocytes in syngeneic HSCT recipients with lymphoma .................48
Figure 4C-D. Multiple vaccinations with tumor cells secreting gp96-Ig increased survival
in T cell replete syngeneic HSCT recipients with lymphoma ...........................................49
Figure 5A. IL-2 administered in vivo after complex with anti-IL-2 mAb effectively
expanded CD8+, but not CD4+, T lymphocytes induced by vaccination with tumor cells
secreting gp96-Ig in lymphoma bearing syngeneic HSCT recipients ...............................50
Figure 5B. IL-2 administered in vivo after complex with anti-IL-2 mAb effectively
expanded tumor-reactive CD8+ T lymphocytes induced by vaccination with tumor cells
secreting gp96-Ig in lymphoma bearing syngeneic HSCT recipients ...............................51
Figure 5C. IL-2 administered in vivo after complex with anti-IL-2 mAb augmented antitumor immunity induced by vaccination with tumor cells secreting gp96-Ig in lymphoma
bearing syngeneic HSCT recipients ...................................................................................52
Figure 5D. IL-2S4B6 in the absence of vaccination markedly expanded CD8+ T cells, but
only elicited a small response by tumor-reactive CD8+ T lymphocytes ............................53
Figure 5E. CD8+ T cells markedly expanded following vaccination and IL-2S4B6 in the
absence of transgenic CD8+ OT-I T lymphocytes resulting in enhanced survival ............54
Figure 5F. IL-2S4B6 in combination with vaccination elicits potent effector CD8+ T cell
response..............................................................................................................................55
Figure 5G. Combined IL-2S4B6 and gp96-Ig vaccination elicited increased CD8+ effector
T cells and enhanced survival in a highly aggressive minimal residual lymphoma model
post-HSCT .........................................................................................................................56
Figure 6A. CD4+ and CD8+ T cell content of purified CD4+ or CD8+ T cells ..................57
Figure 6B. Donor CD8+ T cells efficiently expanded following vaccination and IL-2S4B6
in the absence of donor CD4+ T cells ................................................................................58
Figure 6C. In the absence of donor CD4+ T cells, donor CD8+ T effector cells were not
efficiently generated following vaccine and IL-2S4B6 ........................................................59
Figure 6D. Donor non-CD8+ cells were required for optimal anti-tumor immunity
induced by combination therapy with gp96-Ig secreting tumor cell vaccination and IL-2
cytokine-antibody complexes in syngeneic HSCT recipients with lymphoma .................60
Table 1. Anti-tumor memory response in previously vaccinated HSCT recipients ..........61
Table 1 Figure. Kinetics of tumor growth and survival in previously vaccinated HSCT
recipients challenged with a lethal number of tumor cells ................................................61
v
Figure 7A. IL-2 complex therapy enhanced NK cell numbers in HSCT recipients ..........62
Figure 7B. IL-2 complex therapy enhanced and anti-pathogen immunity in HSCT
recipients ............................................................................................................................63
Figure 8A: Effects of IL-2JES6 ............................................................................................64
Figure 8B. Comparison of IL-2mAb monotherapy ..............................................................65
Figure 8C. Comparison of IL-2mAb in combination with vaccination ...............................66
Table 2: Summary of IL-2/anti-IL-2 complex induced CD8+ T cell responses in the
presence and absence of vaccination with gp96-Ig secreting tumor cells .........................67
Figure 9. Depletion of NK cells in HSCT recipients resulted in diminished tumor-reactive
CD8+ T cell expansion and survival following vaccine and IL-2 ......................................68
vi
LIST OF PUBLICATIONS
Newman, R.G., M.J. Dee, T.R. Malek, E.R. Podack, and R.B. Levy. Heat shock protein
vaccination and targeted IL-2 therapy amplify tumor immunity rapidly following bone
marrow transplantation in mice. Blood 2014 May 8, 123(19): 3045-55, published online
2014 March 31.
Comment in Nature SciBX: Science-Business eXchange 7(17), published online
2014 May 1.
Newman, R.G., D.B. Ross, H. Barreras, S. Herretes, E.R. Podack, K.V. Komanduri, V.L.
Perez, and R.B. Levy. The allure and peril of hematopoietic stem cell transplantation:
overcoming immune challenges to improve success. Immunologic Research 2013
December, 57(1-3): 125-39.
vii
CHAPTER I: Introduction
Section 1: Hematologic Malignancies and Current Treatment
Cancer is the second leading cause of death in the United States, exceeded only
by heart disease, and will account for nearly 25% of all mortality in 2014 [1]. Even
though nearly 1.7 million people will be diagnosed with some form of cancer in 2014 [1],
the overall incidence has been declining yearly since the early 1990s, on average by
about 1.5% per year in the last decade [2]. Malignancies of hematopoietic origin
(leukemia, lymphoma, and myeloma) account for 9% of all new cases of cancer and over
9% of all cancer related mortalities [3]. Cancer is a disease of aging, but hematologic
malignancies, especially leukemia, account for over 40% of all childhood cancer [3]. The
maximal incidence for hematologic malignancies occurs between the ages of 80 and 84,
except for Hodgkin lymphoma, which has a bimodal incidence, with the first maximal
incidence occurring between the ages of 20 and 24 [3]. B cell lineage tumors (e.g., B cell
chronic lymphocytic leukemia (B-CLL), diffuse large B cell lymphoma (DLBCL), and
multiple myeloma) comprise the majority of hematologic malignancies [3], and arise at
different stages of B cell development (Figure A,top) [4]. Similarly, malignancies of T
cell origin (e.g., T cell acute lymphoblastic leukemia (T-ALL), T cell lymphoblastic
lymphoma (T-LL), and T cell CLL) arise at different stages of T cell development and
are associated with certain genetic mutations (Figure A,bottom) [5]. It is estimated that
over 1 million Americans are currently living with or in remission from hematologic
malignancies [3].
Even though five year relative survival rates for patients with hematologic
malignancies are slowly rising due to accurate diagnosis of subtype and improved
1
2
treatment [3], there is still a need for more effective therapies. The first class of
chemotherapeutic agents (i.e., nitrogen mustard, e.g., cyclophosphamide) was
administered to a patient with non-Hodgkin lymphoma over 70 years ago, after
physicians noted lymphoid hypoplasia and myelosuppression in deceased soldiers
exposed to mustard gas during World War I [6]. A few years later, the second class of
chemotherapeutic drugs (i.e., folate analogues, e.g., methotrexate) was used in children
with acute lymphoblastic leukemia, after the observation that folic acid stimulated the
proliferation of these cells in patients [7]. In addition to conventional chemotherapy and
radiation, hematologic malignancies are now being treated with a wide array of targeted
drugs, including kinase inhibitors, histone deacetylase inhibitors, hypomethylating or
demethylating agents,
immunomodulators,
monoclonal antibodies,
antibody-drug
conjugates, and proteasome inhibitors [3]. Recently, June and colleagues have pioneered
the use of adoptive cellular therapy with CD8+ T cells expressing chimeric antigen
receptors targeting CD19 or CD20 [8], which have shown efficacy in patients with B cell
leukemias and lymphomas [9-16]. Patients who are nonresponsive to this arsenal of
therapies might be candidates for hematopoietic stem cell transplantation (HSCT) [3] .
In 2014, it is estimated that myeloma will be diagnosed in over 24,000 people and
cause over 11,000 deaths in the United States [1]. There are almost 90,000 people in the
United States living with or in remission from myeloma [3], and the five year relative
survival rate has significantly increased from 25% in 1975-1977 to 45% in 2003-2009
[1], a time period during which incidence has increased almost 30% [3]. The median age
at diagnosis is 69 years, and interestingly, myeloma rarely occurs under age 45 [3]. Ever
since a landmark prospective randomized study showed that high dose chemotherapy
3
followed by autologous HSCT was superior to conventional chemotherapy [17],
symptomatic myeloma is now treated with induction therapy composed of
dexamethasone (i.e., glucocorticoid, e.g., cortisol) and bortezomib (proteasome inhibitor)
with thalidomide or lenalidomide, followed by autologous HSCT [18]. Individuals not
eligible for HSCT because of advanced age or coexisting conditions receive
chemotherapy typically composed of melphalan (nitrogen mustard), prednisone
(glucocorticoid), thalidomide, and bortezomib [18]. Subsequently, all patients receive
consolidation or maintenance therapy composed of a single agent or combinations of the
aforementioned drugs [18].
Section 2: Hematopoietic Stem Cell Transplantation for Hematologic Malignancies
HSCT was first used to treat inherited anemias or immune deficiencies, but has
now also been applied to benign hematologic disorders, autoimmune diseases, and
tolerance induction for solid organ transplant [19]. Initially, HSCT served as a means of
hematopoietic rescue following high dose chemotherapy and radiation to enable
aggressive tumor cell killing. The first successful HSCT for a cancer patient was
performed by E. Donald Thomas and colleagues at the Fred Hutchinson Cancer Research
Center (FHCRC) in Seattle, WA in 1959, an accomplishment later recognized by his
award of the Nobel Prize in Medicine in 1990. Earlier in the 1950s, several groups
performed pivotal animal studies demonstrating survival of mice exposed to lethal total
body irradiation (TBI) following shielding of the spleen [20] or intravenous infusion of
unfractionated bone marrow cells [21]. The latter was eventually shown to be dependent
on the colonization of recipient bone marrow by transplanted donor cells [22-26]. This
groundbreaking pre-clinical research provided the foundation for subsequent translational
4
studies that first demonstrated the safe infusion of bone marrow cells into cancer patients
receiving radiation and/or chemotherapy with evidence of donor cells in a subset of
recipients suggesting transient engraftment [27].
Autologous HSCT has been primarily used for patients with lymphoma or
myeloma (Figure B) [28]. However, for a brief period from the 1980s to 1990s,
autologous HSCT was assessed in breast cancer patients, and at one point was the
malignancy receiving the most transplants (Figure C) [28]. Stem cell rescue with
autologous HSCT permitted the use of highly aggressive therapy up to twice the standard
non-transplant dose in breast cancer patients [29,30]. Since 1999, 15 randomized clinical
trials compared high dose chemotherapy paired with autologous HSCT to standard of
care regimens for breast cancer patients [29,30]. Unfortunately, meta-analysis of these
breast cancer trials, which included over 6000 patients, found no benefit for autologous
HSCT with regard to overall survival [29,30]. Notably, recipients of autologous HSCT
had increased relapse free survival suggesting anti-tumor activity in breast cancer
[29,30].
Initially, conditioning regimens for HSCT consisted of a single high dose of total
body irradiation (TBI). Conditioning is required to make space for the transplanted stem
cells and promote long term engraftment by deleting T cells that would attack the
transplanted cells, in addition to the tumoricidal effects of the conditioning. Current
conditioning protocols employ fractionated low doses of targeted irradiation and/or drugs
that target tumor cells preferentially over hematopoietic stem cells. Allogeneic HSCT
inherently possesses anti-tumor immunity by the attack of T cells on the tumor, but
carries the serious complication of graft vs host disease and susceptibility to pathogens by
5
the attack of T cells on the non-tumor hematopoietic and non-hematopoietic
compartments. On the contrary, autologous HSCT does not possess the risk of graft vs
host disease because the self T cells do not attack the recipient and has a lower incidence
of infection because of the sparing of some of the hematopoietic compartment, but
superficially lacks anti-tumor immunity because of the self T cells that have been tolerant
to the tumor. Leukemias are generally not treated with autologous HSCT because lower
relapse rates can be achieved with allogeneic HSCT. Similarly, lymphoma and myeloma
are generally not treated with allogeneic HSCT because less graft vs tumor effects have
been observed. Regardless of autologous or allogeneic HSCT, relapse of the primary
disease remains the major cause of mortality in this patient population [28]. Thus, there is
a critical need for more effective therapies in patients receiving HSCT.
Section 3: Vaccination for Hematologic Malignancies following Autologous HSCT
William Coley is credited as the father of immunotherapy for his use of heatkilled Streptococcus pyogenes and Serratia marcescens (Coley toxin) in the 1890s that he
injected directly into tumors. Coley noted regressions following development of fevers in
patients. The first therapeutic cancer vaccine was not approved by the Food and Drug
Administration in the United States until 2010. Sipuleucel-T (Provenge®) is an antigen
presenting cell therapy for metastatic hormone-refractory prostate cancer. The vaccine
consists of autologous dendritic cells obtained by leukapheresis exposed to a fusion
protein of prostatic acid phosphatase, which is expressed by 95% of prostate cancer cells,
and granulocyte-macrophage colony stimulating factor, to mature and activate the
dendritic cells [31]. Meta-analysis of 3 clinical trials comparing Sipuleucel-T to placebo
6
that included over 700 patients revealed a reproducible increase in overall survival for
Sipuleucel-T treated patients [32].
From the 1940s to the 1970s, reports started emerging suggesting that
immunization with viable tumor cells could provide immunity in mice against subsequent
challenge with the same cells [33-37]. For hematologic malignancies, B cell lymphoma
derived Ig idiotype immunization in mice [38] led to clinical trials in lymphoma and
myeloma patients [39-42]. In the 1980s, Srivastava et al. demonstrated that a 96 kDa
protein could complement the immunity induced by immunization with tumor cells [43].
Glycoprotein (gp)96 is the resident endoplasmic reticulum (ER) member of the heat
shock protein (hsp)90 superfamily and is intimately involved in major histocompatibility
(MHC) class I restricted antigen presentation. Once proteins are processed by the
proteasome and their antigenic fragments enter the ER, peptides with appropriate amino
acid sequence are transferred from gp96 to calreticulin to MHC-I before transiting to the
cell surface where peptide is presented. Following necrosis, gp96-antigen complexes are
released from dying cells and can be taken up by antigen presenting cells (APC) in
tissues, which cross-present the chaperoned peptides on MHC-I to specific CD8+ T cells.
APC uptake of gp96 also induces activation and expression of a multitude of cytokines
and chemokines (TNF-α, IL-1β, IL-6, IL-12, GM-CSF, MIP-1α, MCP-1, RANTES, and
NO). Subsequently, Srivastava and colleagues introduced autologous tumor-derived gp96
preparations into patients with cancer, including lymphoma [44,45]. Although immune
correlates were elicited, overall patient survival was relatively unchanged compared with
standard of care regimens [46].
7
In the late 1990s, Podack and colleagues developed a strategy to have cells secrete
gp96 by replacing the ER retention signal (KDEL) with the FC portion of IgG [47].
Vaccination with tumor cell lines transfected to secrete this gp96-Ig molecule induced
CD8+ T lymphocyte and natural killer (NK) cell activation and expansion, leading to
enhancement of anti-tumor (mouse) and anti-viral (monkey) immunity [48-55]. Notably,
the first phase I trial employing a human allogeneic lung cancer cell line engineered to
secrete gp96-Ig was recently completed [56]. Interestingly, ~75% of evaluable patients
displayed CD8+ T cells that secreted IFN-γ when stimulated ex vivo with the vaccine
cells after one, two, or three courses of treatment, and these subjects had a median
survival time (MST) of 16.5 months compared to 4.5 months for non-responders. In
summary, potent vaccination of cancer patients with tumor cells secreting gp96-Ig,
purified gp96, or other heat shock protein preparations is unable to provide generalized
cures as single agent therapies and therefore combining vaccination, which provides the
initial antigenic stimulation, with an adjuvant to enhance this antigen-specific response,
might be necessary to provide sustainable cures to cancer patients.
Section 4: Interleukin-2
T cell growth factor [57], now known as interleukin (IL)-2, was originally cloned
by a group in Japan in 1983 [58], and FDA approved for the treatment of metastatic renal
cell carcinoma and metastatic melanoma in 1992 and 1998, respectively. In mouse
models of melanoma (B16) and fibrosarcoma (MCA-207), administration of IL-2 has
been combined with vaccination [59-64], including following HSCT [65]. The initial
report of IL-2 complexed with anti-IL-2 mAb was published over two decades ago [66].
Complexing IL-2 to anti-IL-2 mAb greatly increases the serum half-life of the protein
8
[66-70]. Boyman et al. demonstrated that a particular anti-mouse IL-2 mAb (clone S4B6)
targeted IL-2 to memory phenotype CD8+ T lymphocytes and NK cells [71], which
express the β (CD122) and γ (CD132) chains of the IL-2 receptor, in the absence of the α
(CD25) chain [72,73]. This ‘targeting’ of IL-2 to principally exclude cells expressing
CD25 [70], reduces a side effect that greatly diminishes the clinical translatability of this
therapy. In high doses, IL-2 administration can cause vascular leak syndrome (VLS,
capillary leak syndrome) with symptoms including increased vascular permeability,
hypotension, pulmonary edema, liver cell damage, and renal failure [74]. Initially, VLS
was thought to be caused by stimulation of NK cells, which can lead to the production of
vasoactive mediators, but a recent report suggests that VLS might be triggered by direct
binding of IL-2 to CD25+ endothelial cells [75]. Various groups have successfully used
IL-2 complexes to enhance vaccination [76,77]. Recently, Levin et al. have engineered an
IL-2 protein with similar binding properties as the IL-2 complex [78]. Others have shown
that IL-2 can enhance endogenous anti-pathogen responses, in a CD8+ T lymphocyte and
NK cell dependent fashion [79-82]. Since infection is the second leading cause of
mortality in HSCT recipients [28], a strategy that combines tumor vaccination and
targeted IL-2 lend merit to use in the early period following HSCT, since it theoretically
will enhance both anti-tumor and anti-pathogen responses.
The FHCRC pioneered the use of IL-2 following autologous HSCT in patients
with hematologic malignancies [83] and early following HSCT for acute myeloid
leukemia (AML) [84] and malignant lymphoma [85]. Similar trials were also being
performed in Minneapolis, MN for acute lymphoid leukemia (ALL) [86] and malignant
lymphoma [87]. The first randomized trial employing recombinant soluble IL-2
9
following HSCT concluded that it did not affect survival of ALL patients using the
selected regimen [88], which was corroborated in a subsequent phase II trial for ALL and
AML [89]. Before moving to a phase III, additional phase I/II trials were necessary since
the source of the IL-2 changed [90,91]; however, subsequent phase III for trials for nonHodgkin lymphoma (NHL) [92] and AML [93] found no effect of IL-2 on patient
survival.
Section 5: Overall Rationale and Hypothesis
Given that heat shock protein vaccination alone generally results in suboptimal
immune stimulation paired with minimal effects on overall survival, and the state of
MRD present in the early post-HSCT period, we reasoned that transplantation with
hematopoietic stem cells supplemented with clinically achievable doses of T cells would
provide the foundation for effective vaccination with tumor cells secreting gp96-Ig and
subsequent augmentation with an adjuvant in the form of IL-2/anti-IL-2 complexes. We
sought to test this hypothesis by developing a model of minimal residual lymphoma
simulating relapse by inoculating mice with tumor immediately following high dose
conditioning, in the form of myeloablative lethal TBI, and autologous HSCT.
Subsequently, we employed a treatment protocol established in non-tumor bearing mice
to investigate if the aforementioned strategy holds merit in lethal pre-clinical models.
Further we established the cellular and molecular requirements of optimal immunity.
10
Figure A. Cellular origin of human
B-cell and T-cell malignancies.
Nature Reviews Cancer 2005
R. Küppers
T-LL
T-ALL
T-CLL
I. Aifantis, E. Raetz, and S. Buonamici
Nature Reviews Immunology 2008
11
B.
C.
CHAPTER II: Heat shock protein vaccination and directed IL-2 therapy amplify tumor
immunity rapidly following bone marrow transplantation in mice
Section 1: Summary
Tumor relapse is the primary cause of mortality in patients with hematologic
cancers following autologous HSCT. Vaccination early post-HSCT can exploit both the
state of lymphopenia and minimal residual disease for generation of anti-tumor immunity
[94-100]. Here, multiple vaccinations utilizing lymphoma cells engineered to secrete the
heat shock protein fusion gp96-Ig within two weeks of T cell replete syngeneic HSCT led
to antigen cross-presentation and increased survival of lymphoma bearing mice. To
enhance vaccine efficacy, interleukin (IL)-2 was directed to predominantly memory
phenotype CD8+ T lymphocytes and natural killer cells via administration bound to antiIL-2 monoclonal antibody clone S4B6 (IL-2S4B6). Combination therapy with gp96-Ig
vaccination and coordinated infusions of IL-2S4B6 resulted in marked prolongation of
median survival time and overall survival, which directly correlated with a ~500%
increase in effector CD8+ T cell numbers. Notably, this dual regimen elicited large
increases in both donor CD8+ T lymphocytes and natural killer cells, but not CD4+ T
lymphocytes, the former two populations essential for both vaccine efficacy and
protection against opportunistic infections post-HSCT. Indeed, IL-2S4B6 treated HSCT
recipients infected with Listeria monocytogenes exhibited decreased bacterial levels.
These pre-clinical studies validate a new strategy particularly well suited to the postHSCT environment, which may augment adaptive and innate immune function in patients
with malignant disease receiving autologous HSCT.
12
13
Section 2: Background
According to CIBMTR, ~80% of mortality after autologous HSCT (2010-2011)
resulted from relapse of primary disease or infection in patients with myeloma,
lymphoma, and leukemia [28]. Multifaceted immunotherapeutic approaches combined
with HSCT for patients with hematopoietic malignancy continue to hold large, but as yet
unfulfilled promise [19]. Such enthusiasm for immune-based strategies rests in part from
the notion that vaccination regimens can be employed early post-HSCT during ‘reboot’
of the immune system to promote efficient anti-tumor and anti-pathogen immunity by
taking advantage of minimal residual disease as well as the state of lymphopenia present
[94-100]. Nevertheless, generating successful protocols early post-HSCT must account
for the relative dearth of T cells as well as the need for a vaccine with appropriate tumor
or pathogen antigens to promote successful immunity.
Heat shock protein gp96 is the resident endoplasmic reticulum protein chaperone
and is intimately involved in MHC-I restricted antigen presentation [43,101-106].
Following necrosis, gp96-peptide complexes are released and can be taken up by local
antigen presenting cells (APC) through CD91 receptor mediated endocytosis leading to
peptide delivery and their efficient activation [107-109]. These APC can therefore crosspresent gp96-chaperoned peptides to CD8+ T lymphocytes inducing their activation,
expansion, and development of effector function [110,111].
The vaccine used in the present studies consisted of tumor cells engineered to
secrete a modified gp96 molecule lacking the ER KDEL retention signal fused to the FC
portion of murine IgG1 (gp96-Ig) [47]. A single vaccination with OVA-expressing cells
secreting gp96-Ig resulted in expansion of endogenous and transgenic OVA257-264-
14
specific CD8+ T cells, that expressed IFN-γ upon stimulation ex vivo and responded more
strongly to a second vaccination in vivo [48]. Notably, gp96-Ig vaccination also
stimulated NK cells and DC, populations hypothesized to contribute to CD8+ T cell
expansion, and required perofrin-1 and IFN-γ, but were independent of FasL [49].
Subsequently, Oizumi and colleagues demonstrated that the system was independent of
CD4+ T cells, CD40L expression, NKT cells, and LN, but required CD80/CD86,
recruited F4/80lo cells, and expanded NK cells and DC at the site of vaccination [50].
Furthermore, it was demonstrated that vaccine-specific CD8+ T cell expansion
following gp96-Ig vaccination was diminished in tumor bearing mice in a tumor antigen
independent manner; however, this could be overcome following multiple vaccinations or
vaccination in the absence of B cells [51]. Schreiber and colleagues then partitioned
responders and non-responders, and determined that enhanced anti-tumor immunity
resulted in increased Th1 and Th17 factors and decreased Th2 factors [52]. Strbo and
colleagues then determined that gp96-Ig vaccination elicited responses in the gut
associated lymphoid tissue and was dependent on the site of vaccination [53]. Moreover,
Strbo and colleagues demonstrated that gp96-Ig vaccines expressing simian
immunodeficiency virus (SIV) proteins were able to elicit polyfunctional CD8+ T cells
specific for multiple SIV antigens in rhesus macaques [54], which resulted in prolonged
survival following challenge with acute SIV infection [55]. Furthermore, the first clinical
trial employing tumor cells secreting gp96-Ig was recently completed in lung cancer
patients, and demonstrated that 75% of vaccinated patients contained CD8+ T cells
expressing IFN-γ following ex vivo stimulation with the vaccine cells, who interestingly
had over 3 fold enhanced survival compared to non-responders [56].
15
IL-2 therapy has demonstrated significant anti-tumor activity in experimental
models of leukemia and has diverse affects following HSCT in part dependent on dose
and time of infusion [112,113]. However, since IL-2 induced expansion of T regulatory
cells (Treg) could inhibit anti-tumor immunity [114], an important advance for use of this
cytokine would be to direct its activity primarily to anti-tumor effector vs Treg cells
[70,71,75]. Notably, several groups have reported that IL-2 conjugated to a particular
anti-IL-2 mAb can augment anti-tumor responses [76,77]. One cytokine-antibody
complex utilizing mAb clone S4B6 (IL-2S4B6), which activates the intermediate affinity
IL-2 receptor (β and γ), was found to stimulate the proliferation of predominately
memory phenotype CD8+ T lymphocytes and NK cells—two populations essential for
optimal gp96-Ig induced anti-tumor responses [49].
The pre-clinical studies described here investigated the efficacy of vaccination
with tumor cells secreting gp96-Ig together with IL-2S4B6 complex in experimental mouse
models of minimal residual lymphoma following syngeneic HSCT. The results obtained
support the notion that the primary effect of gp96-Ig vaccination via antigen crosspresentation early following autologous HSCT was to elicit tumor-reactive CD8+ T cells,
and together with directed IL-2 treatment, markedly augmented effector CD8+ T cell
levels. Global expansion of donor CD8+ T lymphocytes and NK cells, but not CD4+ T
lymphocytes, following administration of this IL-2S4B6 complex contributed to prolonged
survival of lymphoma bearing HSCT recipients as well as augmented anti-pathogen
responsiveness early post-HSCT.
16
Section 3: Methods
Mice. C57BL/6 wild type mice (B6, CD45.2+ CD90.2+), B6-CD45.1+ and B6CD90.1+ congenic strains, and B6-CD80−/−CD86−/− double deficient mice (B7KO) [115]
were obtained from Charles River Laboratories, Taconic Farms, The Jackson Laboratory,
or the National Cancer Institute. B6-OT-I mice [116], provided by M. Bevan, were
backcrossed onto B6-RAG1−/− background [117]. Batf3 deficient (B6-Batf3−/−, Batf3KO)
[118,119] BM was provided by the laboratory of P. Reddy who received animals from K.
Murphy. Perforin-1 deficient mice (B6-Pfp−/−, PrfKO) were generated as described [120].
B6-FoxP3mRFP mice [121] were provided by R. Flavell. Mice were subsequently bred and
maintained under DVR supervision and IACUC approved protocols.
Antibodies and staining. Fluorescent antibodies were purchased from BDBiosciences, eBioscience, BioLegend, or Life Technologies, and used for flow cytometric
analysis: anti-CD4 (RM4-5), anti-CD8α (53-6.7), anti-CD11b (M1/70), anti-CD11c
(N418), anti-CD19 (6D5), anti-CD25 (PC61), anti-CD44 (IM7), anti-CD45.1 (A20), antiCD62L (MEL-14), anti-CD90.1 (OX-7), anti-CD122 (TM-β1), anti-CD127 (A7R34),
anti-F4/80 (BM8), anti-Gr-1 (RB6-8C5), anti-IFN-γ (XMG1.2), anti-KLRG-1 (2F1),
anti-NK1.1 (PK136), anti-TNF-α (MP6-XT22), anti-Vα2 (B20.1), anti-Vβ5.1/5.2 (MR94). To enumerate TCR OVA257-264 (SIINFEKL) reactive CD8+ T cells, 0.5 μg
DimerX−Kb (BD Biosciences) was incubated with 0.33 μg OVA257-264 overnight.
Intracellular cytokine staining was performed using single cell suspensions prepared from
tissues followed by incubation (1.0 x 106 cells/mL) in 10% complete medium with 0.1
nM OVA257-284 for 4-6 h at 37°C, with monensin (GolgiStop, BD-Biosciences) or
brefeldin A (GolgiPlug, BD-Biosciences). Cells were then surface stained, fixed and
17
permeabilized with the FoxP3 staining kit (eBioscience) overnight, followed by
intracellular staining and analysis. Data was acquired on a BD-LSRFortessa, LSRII, or
Accuri-C6 flow cytometer and results analyzed using BD-FACSDiva or CFlow software.
Tumor cell lines. EL-4 lymphoma cells [122] expressing OVA (E.G7) [123],
provided by M. Bevan, were engineered to secrete gp96-Ig (E.G7-gp96-Ig as well as
EL4-gp96-Ig) as described [47]. Cell lines were maintained in Iscove's modified
Dulbecco's medium containing 10% FBS, 1 μg/mL gentamicin, 0.05 mmol/L βmercaptoethanol, and the appropriate antibiotics: G418 (0.4 mg/mL) or L-histidinol (2
mmol/L).
Preparation of donor T cell populations. CD8+ T cells specific for OVA257-264
(OT-I) were purified by negative selection from spleen and LN of B6-OT-I (RAG1−/−)
mice with the CD8+ T Cell Isolation Kit (Miltenyi Biotec) using magnetic separation
(≥95% by flow cytometry). Polyclonal CD4+ and CD8+ T cells were enriched (≥75%)
following B cell depletion (≥90%) from spleen and LN of donor mice using plate bound
goat anti-mouse IgG/M (Millipore) as described [124,125]. Polyclonal CD4+ (≥88%) and
CD8+ (≥90%) T cells were purified by negative selection following B cell depletion.
BM transplantation. BM was isolated from femurs, tibias, and vertebral columns
as described [124,125]. Briefly, T cells were removed by incubation with anti-CD90.2
(HO134), anti-CD4 (RL174), and anti-CD8 (H022) ascites (1:5) with 12% vol/vol LowTox M rabbit complement (Cedarlane Labs) at a final concentration of 25.0 x 106
cells/mL on ice for 15 min followed by 37 °C incubation for 30 min. B6 mice were
conditioned with 9.5 Gy total body irradiation. The following day, 5.0 x 106 congenic B6CD45.1+ T cell depleted (TCD)-BM cells were infused intravenously alone or together
18
with the selected donor T cell populations (0.2 mL RPMI-1640). Mice were maintained
briefly on gentamicin supplemented water.
Tumor cell inoculation into mice used as BM recipients or T cell donors. E.G7
and EL-4 lymphoma cells were isolated from log-phase cultures. One day following BM
transplants, 1.0 x 105 or 5000 cells, respectively, were injected intraperitoneally (0.5 mL
RPMI-1640). Tumor bearing T cell donor mice were inoculated intraperitoneally with 4.0
x 106 E.G7 cells 3 wk before T cell harvest. Some donor mice were injected
intravenously with 5.0 x 105 negatively selected CD8+ OT-I T cells 1 d preceding tumor
inoculation. For subcutaneous tumor challenge experiments, 1 x 106 E.G7 cells were
inoculated (0.1 mL RPMI-1640).
Vaccination. E.G7-gp96-Ig and EL4-gp96-Ig cells were isolated from log-phase
cultures. Two or three days after adoptive transfer of T cells, 1.0 x 107 irradiated (40-120
Gy) cells were injected intraperitoneally (0.5 mL RPMI-1640). Vaccinations were
repeated on subsequent days as indicated.
IL-2 cytokine-antibody complex. 1.5 μg recombinant mouse IL-2 (eBioscience)
was incubated with 8 μg anti-mouse IL-2 mAb clone S4B6 (BD-Biosciences) for 15 min
at room temperature (IL-2S4B6) as described [71]. IL-2 complexes were injected
intraperitoneally as indicated (0.5 mL PBS).
NK cell depletion. Anti-NK1.1 mAb clone PK136 was administered
intraperitoneally (0.5mL PBS) 1 d before total body irradiation (200μg) and 3d (100μg),
9d (50μg), and 15d (50μg) post-HSCT.
Bacterial
infection
and
determination
of
CFU.
Recombinant
Listeria
monocytogenes expressing OVA134-387 [79,126], provided by H. Chen, was grown in
19
brain/heart infusion broth. Log-phase growing bacteria (OD560 0.2) were diluted (PBS) to
1.0 x 104 CFU and injected intravenously (0.2 mL HBSS). The number of bacteria
injected was calculated and confirmed by subsequent growth on brain/heart infusion agar
plates. Seven days post-infection, single cell suspensions of spleens were prepared by
isolation and disruption using a glass stopper on 0.2 μm screens in 0.05% Triton X-100.
CFU were determined by serial dilutions after incubation for 18 h at 37 °C on brain/heart
infusion agar plates.
Statistical analysis. Paired comparisons performed using t test, multiple analyses
performed using one-way or two-way ANOVA as applicable, and survival analyses
performed using log-rank test: *p ≤ 0.05, **p ≤ 0.01, ***p≤0.001, ****p≤0.0001.
Section 4: Results
CD8+ T cell response elicited following vaccination with tumor cells secreting
gp96-Ig in syngeneic HSCT recipients. To assess the immune consequences of
administering gp96-Ig secreting tumor cells early following autologous HSCT, B6 mice
were conditioned (9.5 Gy) and transplanted with B6-CD45.1+ TCD-BM cells. To monitor
a tumor-reactive CD8+ T cell population, TCD-BM was supplemented with 1.0 x 106
CD8+ T cells specific for OVA257-264 (OT-I). Following a single intraperitoneal
vaccination with 1.0 x 107 EL-4 lymphoma cells (120 Gy) expressing OVA (E.G7)
secreting gp96-Ig (E.G7-gp96-Ig), large numbers of macrophages (F4/80hi CD11bhi Gr1lo) and inflammatory monocytes (Gr-1hi CD11bmid F4/80lo) [127,128] rapidly
accumulated at the vaccine site (2 d post-vaccination), comprising ~90% of its entire
cellularity (Figure 1A). Initially, these myeloid cells were of recipient origin, but were
rapidly replaced by their donor counterparts over the course of the immune response
20
(within 1 wk, data not shown). Interestingly, at 2 d post-vaccination, tumor-reactive
CD8+ T lymphocytes could not be detected at the vaccine site (Figure 1A); however,
within 24 h, elevated levels were observed in the blood (data not shown) and maximum
numbers were reached in the peritoneal cavity 5 d following a single vaccination (Figure
1B). At this time, large numbers of macrophages could still be enumerated in this
compartment, together with lower numbers of inflammatory monocytes (Figure 1B).
Notably, vaccination with parental E.G7 cells containing endogenous gp96 did not induce
these cellular responses (Figure 1A-B).
Syngeneic HSCT recipients vaccinated a second time (6 d following the first
vaccination) with tumor cells secreting gp96-Ig contained similar numbers of tumorreactive CD8+ T cells at the vaccine site as observed after a single vaccination, and
elevated numbers of these cells were now detected in the spleen (Figure 1C).
Proliferative signals engendered by lymphopenia diminish over time in myeloablatively
conditioned mice [99,129]. We found that vaccination could be delayed 1-2 wk postHSCT and similar levels of tumor-reactive CD8+ T lymphocytes were observed at the
vaccine site 5 d following vaccination (Figure 1D). CD8+ T cells introduced into a
lymphopenic environment assume a central-memory phenotype (CD62L+ CD44+, Figure
1E) [72,73,130-132]. Following a single vaccination with gp96-Ig secreting tumor cells,
≥80% of tumor-reactive CD8+ T cells transitioned to effector cells (CD62L− CD44+,
Figure 1E). Peptide stimulation of these cells ex vivo corroborated this effector
phenotype since ≥60% expressed IFN-γ (Figure 1F). In non-transplanted mice, gp96 has
been reported not to induce stimulation of CD4+ T cells [47,101], and we detected no
appreciable tumor-reactive (OVA323-339) CD4+ T cell (OT-II) expansion following
21
transfer of 1.0 x 106 CD4+ OT-II T cells and a single vaccination with tumor cells
secreting gp96-Ig in syngeneic HSCT recipients (data not shown).
To determine if B7 costimulation was required for antigen-specific CD8+ T cell
expansion induced by gp96-Ig vaccination in HSCT recipients as well as the source of
this signal regarding APC, transplants were performed utilizing donor and/or recipient
mice deficient in CD80/CD86 (B7KO). When vaccine was administered 2 d post-HSCT,
donor and recipient B7 expressing cells were both found to contribute to overall tumorreactive CD8+ T lymphocyte expansion (Figure 2A). Strikingly, when vaccination was
delayed 1 wk post-HSCT, only B7 expression by donor cells contributed to the response,
since such expansion was not observed when B7 deficient BM cells were transplanted
into WT hosts (Figure 2B).
We next wanted to formally address if cross-presentation was taking place early
post-HSCT since gp96 is thought to function by shuttling antigen into the MHC-I
pathway inside the APC. To address this, TCD-BM from Batf3 deficient mice
(Batf3KO), which lack CD11c+ CD8α+ cross-presenting dendritic cells [118,119], was
used for transplantation (Figure 2C,top). These recipients were unable to expand tumorreactive CD8+ T cells following gp96-Ig vaccination (Figure 2C,bottom). Our group
reported that perforin-1 was required for tumor-reactive CD8+ T lymphocyte expansion
induced by vaccination with tumor cells secreting gp96-Ig in non-transplanted mice [49].
Using either donors or recipients deficient in perforin-1 (PrfKO), transplants determined
that perforin-1 was also required following HSCT, and interestingly, donor or recipient
derived perforin-1 was sufficient to support this expansion (Figure 2D).
22
Effectiveness of gp96-Ig secreting tumor cell vaccination in a pre-clinical
minimal residual lymphoma syngeneic HSCT model. Similar to what occurs in
autologous clinical transplants, T cells were obtained for these experiments from
syngeneic donor mice bearing tumor. Accordingly, TCD-BM (as in Figure 1) was
supplemented with 2.0 x 106 enriched CD4+ and CD8+ T lymphocytes from tumor
bearing B6-CD90.1+ donors. To enable CD8+ tumor-reactive T cell monitoring, some
tumor bearing T cell donors were infused intravenously with 5.0 x 105 CD8+ OT-I T cells
1 d preceding tumor inoculation. Three weeks later, based upon TCR Vαβ analysis, we
calculated that ~1000 transgenic tumor-reactive CD8+ T lymphocytes were present with
the 2.0 x 106 donor T cell inoculum when such tumor bearing animals were employed
(Figure 3A). Two days post-HSCT, B6 recipients were vaccinated intraperitoneally with
1.0 x 107 gp96-Ig secreting tumor cells (40 Gy) and repeated every 3 d (for a total of 5
vaccinations). Rapid and potent tumor-reactive CD8+ T lymphocyte expansion was
identified within the first 3 wk post-HSCT (Figure 3B). Five days following the third
vaccination (at the peak of the response, Figure 3B), tumor-reactive CD8+ T cells were
identified at the vaccine site (>1000x the total input number), spleen (>5000x), and
draining LN (>500x, Figure 3C). Following peptide stimulation ex vivo, a majority of
these lymphocytes from the vaccine site exhibited polyfunctional activity [133], coproducing IFN-γ and TNF-α (Figure 3D). Repeated vaccinations with parental E.G7 cells
again (Figure 1B-C,E-F) failed to induce appreciable expansion or responsiveness greater
than that resulting from their presence in lymphopenic conditions alone (Figure 3B-D).
Next, to simulate tumor relapse post-transplant, we developed a minimal residual
lymphoma model based off the work of Korngold and colleagues [134] in which
23
recipients were inoculated intraperitoneally with 1.0 x 105 viable E.G7 lymphoma cells 1
d following HSCT. This resulted in 100% lethality with a median survival time (MST) of
1 mo, regardless of donor T cell source (tumor naive or tumor bearing donors, Figure
4A,left). Higher doses of T cells (>2.0 x 106) resulted in <100% lethality (Figure
4A,right). In this minimal residual lymphoma model, similar (Figure 3B) tumor-reactive
CD8+ T cell expansion was observed following multiple gp96-Ig vaccinations (Figure
4B). These responses correlated with a significant MST extension and increased overall
survival (20% survived >100 d post-HSCT, Figure 4C). Notably, enhanced MST was
dependent on transplant of donor T cells since vaccination failed to prolong survival in
recipients of BM only lacking donor T cells (Figure 4C). Vaccination with parental E.G7
cells again (Figure 3B) failed to affect expansion or survival (Figure 4B-C) and the
source of donor T cells (tumor naive or tumor bearing donors) again (Figure 4A) did not
alter the survival advantage engendered by gp96-Ig vaccination (Figure 4D).
Directed IL-2 augmented CD8+ T lymphocyte expansion and markedly enhanced
survival of lymphoma bearing HSCT recipients. The initial studies demonstrated that
vaccination with gp96-Ig secreting tumor cells could stimulate tumor-reactive CD8+ T
cells and extend survival. To attempt to expand tumor-reactive CD8+ T cells IL-2 was
introduced following vaccination. Recipient mice were transplanted (as in Figure 4) and
IL-2S4B6 was injected i.p. 1 d following each vaccination. We first observed an increase in
total CD8+ T lymphocyte expansion in the peripheral blood and in tissues of HSCT
recipients (Figure 5A,left and data not shown). However, IL-2S4B6 administration only
marginally affected overall CD4+ T lymphocyte (Figure 5A,right) and notably CD4+
FoxP3+ Treg levels (Figure 5A,bottom). Analysis of the vaccine site, spleen, and LN
24
compartments (2 wk post-HSCT at the peak of the response, Figure 5B,left) following
vaccination in combination with IL-2S4B6 demonstrated >30,000x expansion of CD8+ OTI T lymphocytes had occurred over the input number of ~1000, reaching greater than
>30.0 x 106 cells (Figure 5B,right). Consistent with such expansion, this combinatorial
regimen (gp96-Ig vaccination and IL-2S4B6) also resulted in a striking increase in MST
(>100 d post-HSCT) and overall survival (60%, Figure 5C,bottom). In contrast,
administration of the same dose and schedule of unbound IL-2 (not complexed to mAb)
together with vaccination exhibited no effect on overall CD8+ T lymphocyte expansion
(Figure 5C,left) or CD8+ OT-I T cell levels (Figure 5C,right). Importantly, this did not
result in an increase in MST or overall survival compared to vaccination alone (Figure
5C,bottom). Furthermore, to elicit marked expansion of CD8+ OT-I T cells, IL-2S4B6
must be combined with vaccination since although monotherapy with IL-2S4B6 induced
strong total CD8+ T cell expansion (Figure 5D,left), IL-2S4B6 alone only elicited a small
response by CD8+ OT-I T lymphocytes present (Figure 5D,right). Not surprisingly, this
resulted in similar MST and overall survival compared to vaccination alone (Figure
5D,bottom). Notably, IL-2S4B6 treatment in combination with parental E.G7 cells not
secreting gp96-Ig (“weak” vaccine) elicited only a modest response by CD8+ OT-I T
cells above that of IL-2S4B6 alone (Figure 5D,right).
Importantly, MST and overall survival, as well as total CD8+ T cell expansion in
response to IL-2S4B6, were independent of transgenic OT-I presence (Figure 5E,right
and 5E,left). Notably, in vaccinated recipients not receiving OT-I cells, IL-2S4B6 elicited
enhanced levels of CD8+ T effector cells (Figure 5F,left), which has been described with
other vaccination strategies [135]. To determine if tumor-reactive non-TCR transgenic
25
CD8+ T cells were elicited following gp96-Ig vaccination, DimerX−Kb (mAb with MHCI fused to heavy chain after variable region) loaded with peptide was utilized to detect
OVA257-264 specific cells. Importantly, endogenous CD8+ T lymphocytes specific for
OVA257-264 were readily apparent only in recipients of gp96-Ig vaccination, while a
markedly increased CD8+ effector T cell response was only elicited following
vaccination together with IL-2S4B6 treatment (Figure 5F,right).
To test the vaccine strategy using a system without OVA, a highly lethal minimal
residual lymphoma model employing intraperitoneal inoculation of 5000 EL-4 lymphoma
cells 1 d post-HSCT was employed [136,137]. The results illustrated that EL4-gp96-Ig
vaccination in combination with IL-2S4B6 significantly increased CD8+ effector T cells
levels (Figure 5G,left) and extended MST compared to monotherapy with either reagent
(Figure 5G,right).
Moreover, the MST of the individual
treatments were
indistinguishable from non-vaccinated mice (Figure 5G,right).
To identify which donor leukocyte populations were required for augmented antitumor immunity following vaccine and IL-2 therapy, donor TCD-BM was supplemented
with purified CD8+ or CD4+ T cells in the absence of OT-I (Figure 6A). Notably, donor
CD8+ T cells expanded following vaccine and IL-2S4B6 treatment in the absence of donor
CD4+ T cells (Figure 6B,left and 6B,right). However, a significantly smaller portion of
these expanded donor CD8+ T cells exhibited an effector cell phenotype (CD62L−
CD44+, Figure 6C). These recipients also trended to shortened MST and overall survival
compared to recipients of CD4+ and CD8+ T cells (Figure 6D). Importantly, recipients of
purified CD4+ T cells treated with gp96-Ig vaccination and IL-2S4B6 displayed no survival
benefit compared to non-vaccinated recipients (Figure 6D).
26
To assess for a memory response in previously vaccinated HSCT recipients,
surviving mice (>100 d post-HSCT, Figure 4-6) were challenged with a lethal number
(1.0 x 106) of E.G7 lymphoma cells subcutaneously to determine if tumor-specific
memory had been generated. Surviving HSCT recipients (n = 20) previously vaccinated
with tumor cells secreting gp96-Ig alone (n = 6) or in combination with IL-2S4B6 (n = 14)
exhibited a rejection rate of 90% (Table 1). The animals that were not protected
following re-challenge (2/20) were greater than 1 y of age and as expected, untreated,
non-transplanted mice were also not protected (9/9, see Table 1 Figure,left for kinetics
of tumor growth and Table 1 Figure,right for recipient survival). Interestingly,
recipients transplanted with purified CD8+ T cells (n = 2) also rejected this subcutaneous
tumor challenge (data not shown).
Administration of directed IL-2 enhanced innate and adaptive immunity in the
early post-HSCT period. The vaccine site was analyzed following 3 treatments with IL2S4B6 (2 wk post-HSCT at the peak of the CD8+ T cell response, Figure 5A) to address the
effect of directed IL-2 on NK cells post-transplant [138]. Notably, NK cell numbers were
significantly increased at the site of vaccination (>20x) compared with non-IL-2 treated
recipients (Figure 7A). To determine if the IL-2 induced increases in NK cells and CD8+
T lymphocytes altered anti-pathogen immunity early following HSCT, recombinant
Listeria monocytogenes was inoculated intravenously into mice 2 wk post-HSCT. One
wk post-inoculation, untreated non-transplanted mice and HSCT recipients contained a
similar number of CFU in the spleen (Figure 7B,right). IL-2S4B6 treated HSCT recipients
exhibited elevated CD8+ T cell levels following 4 IL-2S4B6 infusions (Figure 7B,left) and
significantly fewer CFU numbers 1 wk later (Figure 7B,right).
27
Section 5: Discussion
Enhancing diminished immune function is a major challenge following regimens
involving aggressive therapy and autologous HSCT particularly in patients with
hematologic malignancies. An ideal approach would simultaneously elicit rapid and
potent tumor-specific responses, while successfully augmenting overall anti-pathogen
immunity. The principal objective of these experiments was to evaluate the efficacy of a
tumor cell vaccine combined with IL-2 in experimental models of minimal residual
lymphoma during the early period following myeloablative conditioning and syngeneic
HSCT. The results demonstrated that vaccination with tumor cells engineered to secrete
gp96-Ig initiated within a few days post-HSCT induced the activation, expansion, and
functional competence of tumor-reactive CD8+ OT-I T lymphocytes. Notably,
administration of IL-2 pre-bound to an anti-IL-2 mAb clone S4B6 alone or together with
vaccination, led to rapid and extensive expansion of total CD8+ T lymphocytes and NK
cells, and augmented the generation of anti-pathogen (Listeria monocytogenes)
responses. Overall, this strategy of coupling directed IL-2 to heat shock protein
vaccination induced remarkable expansion of effector CD8+ T lymphocytes and led to
dramatic increases in MST and overall survival in HSCT recipients as well as the
generation of long lived anti-tumor memory.
Clinical studies have demonstrated that tumor-specific CD8+ T cell responses can
be elicited in myeloma patients within 1-2 mo following autologous HSCT [139].
Responses in these patients were generated employing multi-epitope predicted tumor
antigen vaccines against hTERT and survivin. Following re-infusion of autologous T
cells 2 d post-HSCT and multiple vaccinations beginning at 2 wk, ~1/3 of patients
28
receiving a prime-boost regimen exhibited specific responses to hTERT and survivin. We
sought to transfer T cells from tumor bearing donors, but utilized a multi-epitope vaccine
in the early post-HSCT period to induce robust and specific immunity [140]. Our strategy
differed as it did not employ ex vivo stimulation, but included IL-2 together with a tumor
vaccine in vivo to induce massive CD8+ T cell expansion early post-HSCT. In contrast to
clinical studies in which patients apparently required vaccination prior to T cell collection
and HSCT to subsequently elicit responses to vaccine antigens [141,142], the inoculum
employed here was obtained from donor mice bearing tumor, but they were not
vaccinated. The present study demonstrated that vaccination was absolutely required to
elicit expansion of an endogenous antigen-specific T cell clone (Figure 5F,right).
Nonetheless, the response of this individual non-TCR transgenic tumor-reactive CD8+ T
cell population cannot account for the markedly increased survival observed in recipients
of the combinatorial strategy described here. We hypothesize that additional tumorspecific clones – which cannot be directly monitored – resulting from the multi-epitope
nature of the heat shock protein vaccine, must contribute to the increased survival
observed in recipients of vaccine and IL-2. Based on the enormous increase in CD8+ T
effector cell numbers following combination treatment (Figure 5F,right), we speculate
this response includes low TCR affinity clones that require strong stimulating signals and
can be expanded in the presence of IL-2S4B6.
There have been few pre-clinical reports concerning therapeutic anti-tumor
vaccination in myeloablative models of syngeneic HSCT supplemented with T cells. In
contrast to the multipronged vaccination strategy employed here, those studies required
tumor cell vaccination after de novo thymic T cell poiesis following transplant, higher
29
doses of donor T cells (>2.0 x 106), or presensitization of donor T cells with the tumor
vaccine [143-145]. One objective of this research was to rapidly elicit T cell responses by
transplanted donor cells prior to de novo production of thymic derived T cells.
Importantly, expansion of donor tumor-reactive CD8+, but not CD4+, T cells was
observed following gp96-Ig vaccination alone or together with directed IL-2 within 10 d
(Figure 4B and 5B,left). However, only this combination strategy induced a marked
increase in overall CD8+ T effector cell numbers (~500%) ≤2 wk post-HSCT (Figure
5F,right). This time frame is prior to production and emigration of new thymic derived T
cells, which takes place 2-3 wk post-HSCT [146]. Together with the observation that
gp96-Ig vaccination alone or together with IL-2S4B6 did not evoke protective immunity in
recipients receiving TCD-BM grafts (Figure 4C) or purified CD4+ T cells (Figure 6D),
respectively, we conclude that the vaccination regimens here expanded transplanted
donor CD8+ T cells very early following HSCT, paralleling clinical findings requiring the
addition of large numbers of T cells to provide detectable immunity early post-HSCT
[141].
Few pre-clinical studies have incorporated heat shock protein preparations into
vaccine strategies for treating hematologic malignancies, including myeloma [147,148].
In the context of experimental syngeneic HSCT, multiple vaccinations with purified
syngeneic lymphoma derived gp96 resulted in increased survival in a model of minimal
residual lymphoma [149-151]. First, 2-4x the quantity of gp96 was required perhaps
because our system secreted 1.0 μg/vaccination. Second, the first vaccination was
administered 2 wk post-HSCT, with subsequent vaccinations after the emergence of
30
thymic derived T cells perhaps because the authors could not elicit a strong enough
response with vaccination alone early following HSCT.
Notably, in our immediate vaccination strategy, although recipient APC initially
contributed to eliciting tumor-reactive CD8+ T cells (Figure 2A), the response rapidly
became singularly dependent on donor B7 expressing APC (Figure 2B), which included a
critical CD11c+ CD8α+ cross-presenting DC population (Figure 2C), and required
perforin-1 (Figure 2D). Therefore, enriched donor T cells plus CD34+ progenitors may
not be optimal for use with this vaccine strategy. The role of perforin-1 remains obscure,
but could involve NK-APC interactions early during the response [152-154].
The initial use of IL-2 in combination with vaccination or complexed to an antiIL-2 mAb in the 1990s reported increased CD8+ T lymphocyte proliferation, NK cell
activity, and enhanced anti-tumor immunity in vivo [59,66,67]. The transplant system
employed here was uniquely suitable for IL-2S4B6 complex usage since virtually all CD8+
T cells transplanted into lymphopenic hosts assume a central-memory phenotype
(CD62L+ CD44+ CD122+ CD127+ KLRG-1−, data not shown) and express IL-2 receptor
β and γ chains presumably rendering these donor CD8+ T cells sensitive to IL-2S4B6
stimulation [72,73]. Boyman and colleagues demonstrated rapid CD8+ T cell expansion
following daily (1 wk) administration of IL-2S4B6 post-HSCT [71]. Similarly, in the
transplant model employed here, we detected virtually no change in CD4+ T cell and
Treg levels (Figure 5A,right and 5A,bottom), indicating that under these conditions, IL2S4B6 complex most efficiently targeted transplanted donor CD8+ T cells. Notably, our
data illustrated that vaccination increased the frequency and numbers of tumor-reactive
31
CD8+ OT-I T cells and subsequently IL-2S4B6 markedly amplified CD8+ effector T cell
levels, which directly correlated with the observed increases in MST and overall survival.
Using an independent, highly lethal model of minimal residual lymphoma,
increased MST was only observed following vaccination and IL-2 therapy (Figure 5G)
further supporting the necessity for the combination. Studies have also demonstrated that
human IL-2 complexed to human anti-IL-2 mAb effectively expanded murine CD8+ T
cells and could enhance anti-tumor immunity [70,71,75]. Interestingly, Levin et al. have
engineered human IL-2 to possess binding properties similar to those when natural IL-2
is complexed to anti-IL-2 mAb [78]. These human IL-2 complexes, ‘super-IL-2,’ and
new IL-2 fusion products may therefore provide next generation reagents to implement
and clinically test the regimen developed here. Similar to IL-2/anti-IL-2 complexes, IL15 can be complexed with the α chain of the IL-15 receptor (IL-15Rα) leading to
enhanced anti-tumor immunity [155,156].
In addition to CD8+ T cells, the individual capacities of gp96 and IL-2 to activate
APC and NK cells, respectively, is well documented [66,107,108,138] and therefore
CD4+ T cell presence may not be required for post-HSCT vaccination. IL-2S4B6 treatment
following transplant with purified CD8+ T cells (Figure 6A and 6B,right) resulted in
marked donor CD8+ T cell expansion (Figure 6B,left). However, there were decreased
levels of effector (CD62L− CD44+) CD8+ T cells (Figure 6C), which might have
contributed to the reduced survival observed in these recipients (Figure 6D). These
findings support the notion that donor CD4+ T cell licensing of APC is unnecessary for
this CD8+ T cell expansion and purified donor CD4+ T cells failed to provide detectable
anti-tumor immunity following vaccine and IL-2 treatment.
32
We observed potent NK cell expansion following vaccination and IL-2S4B6
infusion (Figure 7A), a population critical for gp96-Ig based strategies [49]. Thus, we
posit IL-2S4B6 enhanced vaccine efficacy in both an indirect and direct fashion via NK
cell and tumor-reactive CD8+ T lymphocyte expansion. In addition to relapse of primary
disease, infection is the second leading cause of mortality in HSCT recipients [28]. Both
CD8+ T lymphocytes and NK cells are known to contribute to immunity against Listeria
monocytogenes [79-81,126,157,158]. Consistent with this, we found that IL-2S4B6 treated
HSCT recipients, which contain increased CD8+ T lymphocyte and NK cell numbers
early post-HSCT, displayed diminished splenic bacterial levels (Figure 7B,right). This is
the first demonstration we are aware of reporting IL-2 enhanced anti-pathogen immunity
early following myeloablative conditioning and syngeneic HSCT. In models of persistent
viral infection, IL-2 has been shown to enhance CD8+ T cell levels and reduce viral
burden in non-transplanted mice infected with lymphocytic choriomeningitis virus
(LCMV) [159] and murine γ-herpesvirus 68 (MHV-68) [160].
Srivastava and colleagues introduced autologous tumor-derived gp96 preparations
into patients with cancer, including lymphoma [44,45]. Although immune correlates were
elicited, overall patient survival was relatively unchanged compared with standard of care
regimens [46]. Notably, the first successful phase I trial employing a human allogeneic
lung cancer cell line engineered to secrete gp96-Ig was recently completed [56].
Interestingly, ~75% of evaluable patients displayed CD8+ IFN-γ responses from PBMC
against the vaccine cells ex vivo, and these subjects had a MST of 16.5 mo vs 4.5 mo for
non-responders. The present findings support the notion that a more effective
combinatorial strategy can first activate and then expand tumor-reactive CD8+ T cells.
33
Based on the findings presented here, the tumor cell secreted heat shock protein vaccine
and directed IL-2 regimen developed in the pre-clinical model here represents a
promising strategy with translational applications for patients with hematologic cancers.
In addition to IL-2S4B6, IL-2 can be complexed with other anti-mouse IL-2 mAb
such as clone JES6-1A12 (IL-2JES6), which has been shown to efficiently stimulate the
proliferation of cells expressing IL-2 receptor α (CD25) including CD4+ FoxP3+ Treg and
recently activated CD8+ T cells [69-71,75,161]. Experiments were performed to compare
IL-2JES6 to IL-2S4B6 as monotherapies and in combination with vaccination. In contrast to
IL-2S4B6, CD8+ T lymphocytes only expanded following IL-2JES6 treatment in
combination with vaccination, but not as a monotherapy (Figure 8A,left). IL-2JES6 treated
vaccinated mice also displayed elevated levels of tumor-specific CD8+ OT-I T cells
(Figure 8A,right), which was consistent with an enhancement of recipient survival
(Figure 8A,bottom). Another difference between IL-2S4B6 and IL-2JES6 was that
monotherapy with the latter was able to induce tumor-specific CD8+ OT-I T cells,
although only in a subset of animals (3/5), with delayed kinetics compared to vaccination
alone (Figure 8A,right). Monotherapy (Figure 8B) and combination therapy (Figure
8C) are displayed in the same graphs for comparison purposes. Note that there are subtle
differences in magnitude and kinetics of CD8+ T cell responses (total and tumor-specific
OT-I, Table 2), but overall recipient survival is similar comparing either complex alone
or in combination with vaccination.
34
Section 6: Future Directions
We observed reduced activation of donor CD8+ T lymphocytes and shortened
MST and overall survival in tumor bearing syngeneic HSCT recipients transplanted with
purified CD8+ T cells treated with gp96-Ig vaccination and IL-2 complex. To determine
what non-CD8+ cell population contained in the donor T cell inoculum was required for
optimal donor CD8+ T lymphocyte activation, MST, and overall survival in recipients of
enriched CD4+ and CD8+ T cells, the next set of experiments could systematically
remove either donor CD4+ T lymphocytes or NK cells. In a preliminary experiment, mice
were transplanted with either enriched CD8+ T cells (CD4+ depleted) or purified CD4+
and CD8+ T cells (NK, etc., depleted). In both groups, MST and overall survival were
similar to recipients of enriched CD4+ and CD8+ T cells, suggesting that CD4+ T cells
and another non-CD8+ cell population (most likely NK cells) were both contributing to
optimal tumor immunity. Interestingly, it appeared that CD4+ T cells were more
important for CD8+ effector T cell generation as recipients of purified CD4+ and CD8+ T
cells (NK depleted) were more similar to recipients of enriched CD4+ and CD8+ T cells,
whereas recipients of enriched CD8+ T cells (CD4 depleted) were more similar to
recipients of purified CD8+ T cells.
To examine the role of NK cells in our minimal residual lymphoma model,
transplant recipients treated with vaccine and IL-2S4B6 were administered anti-NK1.1
mAb clone PK136 the day before conditioning and every 5-6 d for a total of 4 injections
spanning the vaccine protocol. NK cells were depleted for at least 3 wk post-HSCT
(Figure 9,left). Interestingly, these mice displayed delayed kinetics of and reduced levels
of endogenous tumor-reactive CD8+ T cell expansion compared to similarly treated
35
recipients with an intact NK cell compartment (Figure 9,right), which might have
contributed to reduced MST and overall survival (Figure 9,bottom). In order to remove
NK cells only from the donor T cell inoculum, T cells would be enriched from IL-15
deficient mice, which lack NK cells [162], and then CD4+ T cells would be depleted. This
would be compared to recipients of similarly treated cells from wild type mice along with
purified CD8+ T cells from wild type mice.
To further address how CD4+ T cells contributed to tumor immunity in HSCT
recipients of purified CD4+ and CD8+ T cells, purified CD4+ T cells will be obtained
from CD40L or IL-2 deficient mice and mixed with purified CD8+ T cells from wild type
mice. This will be compared to recipients of purified CD4+ and CD8+ T cells from wild
type mice along with recipients of purified CD8+ T cells from wild type mice.
To identify which functional molecules donor CD8+ T cells employed for optimal
tumor immunity, purified CD8+ T cells will be obtained from perforin-1 or granzyme
deficient mice and mixed with CD8+ depleted cells from wild type mice. These groups
will be compared to recipients of enriched CD4+ and CD8+ T cells from wild type mice
along with recipients of purified CD8+ T cells from wild type mice.
Proposed model for gp96-Ig secreting tumor cell vaccination and IL-2S4B6 therapy:
CD8+ T cell
↗
↘
↑
+
+
gp96-Ig
tumor/pathogen
vaccine →
→ CD11c CD8α DC
IL-2S4B6
↖
↗
↓
NK cell
FIGURES AND TABLES
Figure 1A-B. Peritoneal vaccination with tumor cells secreting gp96-Ig in syngeneic
HSCT recipients led to the infiltration of myeloid cells and expansion of tumorreactive CD8+ T lymphocytes in the peritoneal cavity. Conditioned (9.5 Gy) B6 mice
were transplanted with 5.0 x 106 B6-CD45.1+ TCD-BM cells and adoptively transferred
with 1.0 x 106 CD8+ T cells specific for OVA257-264 (OT-I) 5 d later. After a 2 d resting
period, recipients were vaccinated intraperitoneally with irradiated (120 Gy) EL-4
lymphoma cells expressing OVA (E.G7) engineered to secrete gp96-Ig (E.G7-gp96-Ig).
(A) Macrophage (F4/80hi CD11bhi Gr-1lo) and inflammatory monocyte (Gr-1hi CD11bmid
F4/80lo) numbers 2 d following vaccination; n = 9 from pool of 3 experiments. (B)
Tumor-reactive CD8+ T lymphocyte (OT-I: CD8+ CD45.1− Vα2+ Vβ5+) numbers 5 d postvaccination; n = 8 from pool of 3 experiments.
36
37
Figure 1C. A second vaccination with tumor cells secreting gp96-Ig led to
accumulation of tumor-reactive CD8+ T lymphocytes in secondary lymphoid organs.
Mice were transplanted and treated as in Figure 1A and received a 2nd vaccination 13 d
post-HSCT. Tumor-reactive CD8+ T cell numbers 18 d following HSCT; n = 3.
38
Figure 1D. Equivalent expansion of tumor-reactive CD8+ T lymphocytes following
vaccination up to 2 wk post-HSCT. Transplants were performed as in Figure 1A, but
tumor-reactive CD8+ T cells were co-transplanted with TCD-BM, and recipeints were
vaccinated 2, 7, or 14 d following HSCT. Tumor-reactive CD8+ T cell number 5 d postvaccination; n = 3-8 from 3 independent experiments.
39
Figure 1E. Tumor-reactive CD8+ T lymphocytes are activated following vaccination.
Mice were transplanted and treated as in Figure 1A. Tumor-reactive CD8+ T lymphocytes
transitioned from central-memory (TCM, CD62L+ CD44+) to effector cells (Teff, CD62L−
CD44+) at the site of vaccination; representative dot plots (left), n = 5-6 from pool of 2
experiments (right).
40
Figure 1F. Vaccination results in functional tumor-reactive CD8+ T lymphocytes.
Transplants and treatments were performed as in Figure 1A. Vaccination induced highly
IFN-γ+ tumor-reactive CD8+ T cells at the site of vaccination; representative dot plots
(left), n = 4 from representative of 3 experiments (right).
41
Figure 2A-B. B7 costimulation was required for optimal tumor-reactive CD8+ T
lymphocyte expansion induced by vaccination with gp96-Ig secreting tumor cells
following syngeneic HSCT. Transplants were performed as in Figure 1 utilizing mice
deficient in CD80 and CD86 (B7KO) as donors and/or recipients with (A) co-infusion or
(B) delayed infusion of 1.0 x 106 tumor-reactive CD8+ T cells (OT-I) and the peritoneal
cavity was analyzed 5 d following intraperitoneal vaccination with tumor cells secreting
gp96-Ig. (A) Donor and recipient APC contributed to expansion of tumor-reactive CD8+
T cells, co-transplanted with the BM, following vaccination with gp96-Ig secreting tumor
cells; n = 3-9 from pool of 3 experiments. (B) Only donor APC contributed to expansion
of tumor-reactive CD8+ T lymphocytes, infused 5 d post-HSCT, induced by vaccination
with tumor cells secreting gp96-Ig; n = 2-4 from pool of 2 experiments.
42
Figure 2C. Batf3 was required for optimal tumor-reactive CD8+ T lymphocyte
expansion induced by vaccination with gp96-Ig secreting tumor cells following
syngeneic HSCT. Conditioned (9.5 Gy) B6 mice were transplanted with 5.0 x 106 B6
wild-type (B6WT) or Batf3 deficient (Batf3KO) TCD-BM cells and adoptively
transferred with 0.5 x 106 CD8+ T cells specific for OVA257-264 (OT-I) 5 d later. After a 2
d resting period, recipients were vaccinated intraperitoneally with irradiated (40 Gy) EL4 lymphoma cells expressing OVA (E.G7) engineered to secrete gp96-Ig (E.G7-gp96-Ig).
(Top) Loss of CD11blo CD11chi DC at the site of vaccination in recipients of Batf3
deficient TCD-BM receiving gp96-Ig secreting tumor cells. CD11chi CD11blo F4/80lo Gr1lo frequency in the peritoneal cavity; representative dot plots (left), n = 5 (right).
(Bottom) Donor cross-presenting CD11c+ CD8α+ DC were required for optimal
expansion of tumor-reactive CD8+ T cells following vaccination with gp96-Ig secreting
tumor cells; n = 5.
43
Figure 2D. Perforin-1 was required for optimal tumor-reactive CD8+ T lymphocyte
expansion induced by vaccination with gp96-Ig secreting tumor cells following
syngeneic HSCT. Transplants and treatments were performed as in Figure 1 utilizing
mice deficient in perforin-1 (PrfKO) as donors and/or recipients with delayed infusion of
1.0 x 106 tumor-reactive CD8+ T cells (OT-I) and the peritoneal cavity was analyzed 5 d
following intraperitoneal vaccination with tumor cells secreting gp96-Ig. (D) Perforin-1
could be supplied by either donor or recipient cells for optimal expansion of tumorreactive CD8+ T lymphocytes following vaccination with tumor cells secreting gp96-Ig; n
= 2-4 from pool of 2 experiments.
44
Figure 3A. CD4+ and CD8+ splenic and LN T cells from a typical tumor bearing
donor mouse. Donor mice were injected 3 wk pre-HSCT with 5.0 x 105 CD8+ OT-I T
lymphocytes intravenously and 4.0 x 106 E.G7 lymphoma cells intraperitoneally and bear
progressively growing tumors. Tumor was only detectable at the site of injection
(peritoneal cavity) at this time. Total events analyzed represented 2.5 x 106 viable cells
and CD8+ OT-I T lymphocytes were clearly identified (1000 CD8+ CD90.1− Vα2+ Vβ5+ /
1.0 x 106 CD8+ CD90.1+). 2.0 x 106 CD4+ and CD8+ T lymphocytes obtained from E.G7
lymphoma bearing B6-CD90.1+ donors contained ~1000 tumor-reactive CD8+ T cells
(OT-I).
45
Figure 3B-C. Tumor-reactive CD8+ T lymphocytes obtained from tumor bearing
donors were expanded following transplantation into syngeneic HSCT recipients
and vaccination with tumor cells secreting gp96-Ig. Conditioned (9.5 Gy) B6
recipients received B6-CD45.1+ TCD-BM cells supplemented with 2.0 x 106 B6-CD90.1+
CD4+ and CD8+ T lymphocytes from Figure 3A. Recipients were vaccinated
intraperitoneally with irradiated (40 Gy) E.G7 cells secreting gp96-Ig 2 d post-HSCT and
repeated every 3 d for a total of 5 vaccinations. (B) Multiple vaccinations with gp96-Ig
secreting tumor cells induced expansion of tumor-reactive CD8+ T cells obtained from
tumor bearing donors; n = 4; ♦: E.G7, ●: E.G7-gp96-Ig. (C) Tumor-reactive CD8+ T
lymphocytes expanded at the vaccine site and other lymphoid tissues 5 d following 3
vaccinations with tumor cells secreting gp96-Ig; n = 2.
46
Figure 3D. Tumor-reactive CD8+ T lymphocytes obtained from tumor bearing
donors were functional following transplantation into syngeneic HSCT recipients
and vaccination with tumor cells secreting gp96-Ig. Mice were transplanted and
treated as in Figure 3B. Vaccination with gp96-Ig secreting tumor cells induced highly
IFN-γ+ and TNF-α+ tumor-reactive CD8+ T cells 5 d following 3 vaccinations;
representative dot plots from n = 2.
47
Figure 4A. Development of minimal residual lymphoma model following syngeneic
HSCT. Transplants were performed as in Figure 3 and recipients were inoculated
intraperitoneally with 1.0 x 105 E.G7 lymphoma cells the following day to simulate
tumor relapse post-HSCT. (Left) Equivalent survival of HSCT recipients with lymphoma
receiving T cells from tumor bearing or tumor naive donors or no T cells; n = 4-5 from 3
independent experiments; x: no vaccine (tumor), +: no vaccine (naive), −: no T cells.
(Right) Increased numbers of T cells (>2.0 x 106) results in <100% lethality; n = 4-5
from 2 independent experiments; x: 2.0 x 106 T cells, +: 5.0 x 106 T cells, −: 10.0 x 106 T
cells.
48
Figure 4B. Multiple vaccinations with tumor cells secreting gp96-Ig expanded
tumor-reactive CD8+ T lymphocytes in syngeneic HSCT recipients with lymphoma.
Transplants and tumor inoculation were performed as in Figure 4A. Two days following
tumor inoculation, recipients were vaccinated intraperitoneally with irradiated (40 Gy)
E.G7 lymphoma cells secreting gp96-Ig and repeated every 3 d for a total of 5
vaccinations; n = 20 from pool of 4 experiments; x: no vaccine, ♦: E.G7, ●: E.G7-gp96Ig.
49
Figure 4C-D. Multiple vaccinations with tumor cells secreting gp96-Ig increased
survival in T cell replete syngeneic HSCT recipients with lymphoma. Transplants,
tumor inoculation, and treatments were performed as in Figure 4B. (C) Vaccination with
tumor cells secreting gp96-Ig led to increased MST and overall survival of lymphoma
bearing HSCT recipients; n = 4-5; x: no vaccine, ♦: E.G7, ●: E.G7-gp96-Ig, ○: E.G7gp96-Ig (no T cells). The ‘no vaccine’ group illustrated here was shown in Figure 4A
with other non-vaccinated groups. (D) Equivalent survival of vaccinated HSCT recipients
receiving T cells from tumor bearing or tumor naive donors; n = 6; ●: E.G7-gp96-Ig
(tumor), ○: E.G7-gp96-Ig (naive).
50
Figure 5A. IL-2 administered in vivo after complex with anti-IL-2 mAb effectively
expanded CD8+, but not CD4+, T lymphocytes induced by vaccination with tumor
cells secreting gp96-Ig in lymphoma bearing syngeneic HSCT recipients.
Transplants, tumor inoculation, and treatments were performed as in Figure 4 and mice
received IL-2S4B6 (1.5 μg IL-2 + 8 μg anti-IL-2 mAb clone S4B6) 1 d following each
vaccination. (Left) IL-2S4B6 treatment induced robust expansion of CD8+ T cells in
vaccinated HSCT recipients with lymphoma during the first 3 wk post-HSCT. CD8+ T
cell frequency in the peripheral blood; n = 20 from pool of 4 experiments; x: no vaccine,
♦: E.G7, ●: E.G7-gp96-Ig, ■: E.G7-gp96-Ig + IL-2S4B6. (Right) Systemic CD4+ T cell
levels were unchanged following IL-2S4B6 treatment in syngeneic HSCT recipients. CD4+
T cell frequency in the peripheral blood; n = 10 from pool of 2 experiments; x: no
vaccine, ♦: E.G7, ●: E.G7-gp96-Ig, ■: E.G7-gp96-Ig + IL-2S4B6. (Bottom) Systemic
CD4+ FoxP3+ Treg levels were marginally changed following IL-2S4B6 treatment in
syngeneic HSCT recipients. FoxP3+ frequency within CD4+ T cells in the peripheral
blood utilizing mice expressing red fluorescent protein under control of the FoxP3
promoter as T cell donors; n = pool of 5; x: no vaccine, ♦: E.G7, ●: E.G7-gp96-Ig, ■:
E.G7-gp96-Ig + IL-2S4B6.
51
Figure 5B. IL-2 administered in vivo after complex with anti-IL-2 mAb effectively
expanded tumor-reactive CD8+ T lymphocytes induced by vaccination with tumor
cells secreting gp96-Ig in lymphoma bearing syngeneic HSCT recipients.
Transplants, tumor inoculation, and treatments were performed as in Figure 5A. (Left)
Tumor-reactive CD8+ T cells reached maximal levels in the peripheral blood 2 wk postHSCT following vaccination and IL-2 therapy; n = 20 from pool of 4 experiments; x: no
vaccine, ♦: E.G7, ●: E.G7-gp96-Ig, ■: E.G7-gp96-Ig + IL-2S4B6. The ‘no vaccine,’
‘E.G7,’ and ‘E.G7-gp9-Ig’ groups from Figure 4B were included as a reference to
illustrate the kinetics of expansion with vaccination and IL-2S4B6. (Right) Tumor-reactive
CD8+ T lymphocytes markedly expanded at the vaccine site and other lymphoid tissues
of lymphoma bearing HSCT recipients 5 d following 3 treatments with IL-2S4B6 and
gp96-Ig vaccine. Tumor-reactive CD8+ T cell number in the peritoneal cavity, spleen,
and draining LN; n = 2.
52
Figure 5C. IL-2 administered in vivo after complex with anti-IL-2 mAb augmented
anti-tumor immunity induced by vaccination with tumor cells secreting gp96-Ig in
lymphoma bearing syngeneic HSCT recipients. Transplants, tumor inoculation, and
treatments were performed as in Figure 5A. (Left) Non-complexed, unbound IL-2 in
combination with vaccination failed to expand CD8+ T lymphocytes. CD8+ T cell
frequency in the peripheral blood; n = 5; ●: E.G7-gp96-Ig, ♦: E.G7-gp96-Ig + IL-2, ■:
E.G7-gp96-Ig + IL-2S4B6. (Right) Non-complexed, unbound IL-2 in combination with
vaccination failed to expand tumor-reactive CD8+ T lymphocytes. Tumor-reactive CD8+
T cell frequency in the peripheral blood; n = 5. ●: E.G7-gp96-Ig, ♦: E.G7-gp96-Ig + IL-2,
■: E.G7-gp96-Ig + IL-2S4B6. Extended analyses of these CD8+ T lymphocyte frequencies
in mice treated with vaccination and IL-2S4B6 suggested that slightly increased levels
were maintained for at least 6 wk post-HSCT, or 1 mo following cessation of treatment.
(C) Combination therapy with vaccination and IL-2S4B6 increased MST and overall
survival of syngeneic HSCT recipients with lymphoma. Non-complexed, unbound IL-2
in combination with vaccination failed to enhance survival of lymphoma bearing HSCT
recipients; n = 5; ●: E.G7-gp96-Ig, ♦: E.G7-gp96-Ig + IL-2, ■: E.G7-gp96-Ig + IL-2S4B6.
53
Figure 5D. IL-2S4B6 in the absence of vaccination markedly expanded CD8+ T cells,
but only elicited a small response by tumor-reactive CD8+ T lymphocytes.
Transplants, tumor inoculation, and treatments were performed as in Figure 5A. (Left)
IL-2S4B6 in the absence of vaccination markedly expanded CD8+ T cells. CD8+ T cell
frequency in the peripheral blood; n = pool of 5; x: IL-2S4B6, ●: E.G7-gp96-Ig, ■: E.G7gp96-Ig + IL-2S4B6. (Right) IL-2S4B6 in the absence of vaccination only elicited a small
response by tumor-reactive CD8+ T lymphocytes. Tumor-reactive CD8+ T cell frequency
in the peripheral blood; n = pool of 5; x: IL-2S4B6, ♦: E.G7 + IL-2S4B6, ●: E.G7-gp96-Ig,
■: E.G7-gp96-Ig + IL-2S4B6. (Bottom) IL-2S4B6 in the absence of vaccination marginally
enhanced survival of lymphoma bearing HSCT recipients; n = 5; x: IL-2S4B6, ●: E.G7gp96-Ig, ■: E.G7-gp96-Ig + IL-2S4B6.
54
Figure 5E. CD8+ T cells markedly expanded following vaccination and IL-2S4B6 in
the absence of transgenic CD8+ OT-I T lymphocytes resulting in enhanced survival.
Transplants, tumor inoculation, and treatments were performed as in Figure 5A. (Left)
CD8+ T cell frequency in the peripheral blood; n = pool of 5; ●: E.G7-gp96-Ig, ○: E.G7gp96-Ig (no OT-I), ■: E.G7-gp96-Ig + IL-2S4B6, □: E.G7-gp96-Ig + IL-2S4B6 (no OT-I).
The small increase observed in CD8+ T lymphocyte frequency in both groups containing
CD8+ OT-I T cells likely represents the presence of the transgenic CD8+ T cell
population. (Right) MST and overall survival of T cell replete syngeneic HSCT
recipients with lymphoma following vaccination and IL-2S4B6 were independent of
transgenic antigen-specific CD8+ T cell presence; n = 5; ●: E.G7-gp96-Ig, ○: E.G7-gp96Ig (no OT-I), ■: E.G7-gp96-Ig + IL-2S4B6, □: E.G7-gp96-Ig + IL-2S4B6 (no OT-I).
55
Figure 5F. IL-2S4B6 in combination with vaccination elicits potent effector CD8+ T
cell response. Transplants, tumor inoculation, and treatments were performed as in
Figure 5A. (Left) CD8+ Teff (CD62L− CD44+) cell frequency in the peripheral blood; n =
21 from pool of 4 experiments; ●: E.G7-gp96-Ig, ■: E.G7-gp96-Ig + IL-2S4B6. (Right)
CD8+ effector T cell numbers at the vaccine site were markedly increased only following
vaccine and IL-2S4B6 treatment. CD8+ Teff (CD62L− CD44+), including endogenous nonTCR transgenic DimerX−Kb−OVA257-264+, cell numbers in the peritoneal cavity 5 d
following 3 vaccinations; n = 5.
56
Figure 5G. Combined IL-2S4B6 and gp96-Ig vaccination elicited increased CD8+
effector T cells and enhanced survival in a highly aggressive minimal residual
lymphoma model post-HSCT. Transplants were performed as in Figure 5A and
recipients were inoculated with 5000 EL-4 lymphoma cells 1 d post-HSCT. Mice were
vaccinated with EL4-gp96-Ig and received IL-2S4B6 1 d following each vaccination for a
total of 5 treatments. (Left) IL-2S4B6 enhanced CD8+ effector T cell levels induced by
gp96-Ig vaccination. CD8+ Teff (CD62L− CD44+) cell frequency in peripheral blood; n =
5; ●: EL4-gp96-Ig, ■: EL4-gp96-Ig + IL-2S4B6. (Right) Only combined vaccination and
IL-2S4B6 increased survival post-HSCT; n = 5; x: no vaccine, ♦: IL-2S4B6, ●: EL4-gp96-Ig,
■: EL4-gp96-Ig + IL-2S4B6.
57
Figure 6A. CD4+ and CD8+ T cell content of purified CD4+ or CD8+ T cells. CD4+
and CD8+ T cells were purified following B cell depletion using negative selection.
58
Figure 6B. Donor CD8+ T cells efficiently expanded following vaccination and IL2S4B6 in the absence of donor CD4+ T cells. Transplants, tumor inoculation, and
treatments were performed as in Figure 5, however some recipients received BM
supplemented with purified CD4+ (≥88%) or CD8+ (≥90%) T cells. (Left) Donor CD8+ T
cell frequency in the peripheral blood; n = 5; ●: E.G7-gp96-Ig, ○: E.G7-gp96-Ig (Purified
CD8), ■: E.G7-gp96-Ig + IL-2S4B6, □: E.G7-gp96-Ig + IL-2S4B6 (Purified CD8). The
results for ‘E.G7-gp96-Ig + IL-2S4B6’ and ‘E.G7-gp96-Ig + IL-2S4B6 (Purified CD8)’ were
repeated in an independent experiment. (Right) Donor CD4+ T cells were minimally
detectable in recipients of purified CD8+ T cells. CD4+ T cell frequency in the peripheral
blood; n = 5; ●: E.G7-gp96-Ig, ○: E.G7-gp96-Ig (Purified CD8), ■: E.G7-gp96-Ig + IL2S4B6, □: E.G7-gp96-Ig + IL-2S4B6 (Purified CD8). The results for ‘E.G7-gp96-Ig + IL2S4B6’ and ‘E.G7-gp96-Ig + IL-2S4B6 (Purified CD8)’ were repeated in an independent
experiment.
59
Figure 6C. In the absence of donor CD4+ T cells, donor CD8+ T effector cells were
not efficiently generated following vaccine and IL-2S4B6. Transplants, tumor
inoculation, and treatments were performed as in Figure 6B. Donor CD8+ Teff (CD62L−
CD44+) cell frequency in the peripheral blood; n = 5 from representative of 2
experiments; ●: E.G7-gp96-Ig, ■: E.G7-gp96-Ig + IL-2S4B6, □: E.G7-gp96-Ig + IL-2S4B6
(Purified CD8). The results for ‘E.G7-gp96-Ig + IL-2S4B6’ and ‘E.G7-gp96-Ig + IL-2S4B6
(Purified CD8)’ were repeated in an independent experiment.
60
Figure 6D. Donor non-CD8+ cells were required for optimal anti-tumor immunity
induced by combination therapy with gp96-Ig secreting tumor cell vaccination and
IL-2 cytokine-antibody complexes in syngeneic HSCT recipients with lymphoma.
Transplants, tumor inoculation, and treatments were performed as in Figure 6B. Survival
benefit of vaccination and IL-2S4B6 therapy is reduced in lymphoma bearing HSCT
recipients receiving TCD-BM supplemented with purified CD8+ T cells and abolished in
recipients of purified CD4+ T cells; n = 5-10 from pool of 3 experiments; x: no vaccine,
●: E.G7-gp96-Ig, ■: E.G7-gp96-Ig + IL-2S4B6, □: E.G7-gp96-Ig + IL-2S4B6 (Purified
CD8), ◊: E.G7-gp96-Ig + IL-2S4B6 (Purified CD4).
61
Table 1. Anti-tumor memory response in previously vaccinated HSCT recipients.
c
No. with tumor /
Treatment
No. injected
Untreated
Non-transplanted
E.G7-gp96-Iga
BM + T cellsb
E.G7-gp96-Ig + IL-2S4B6
d
e
% with tumor
9/9
100
0/6
0
a
2 / 14
14.3
BM + T cellsb
a
Transplant recipients were ≥100 d post-HSCT (Figure 4-6).
b
≥75% CD4+ and CD8+; ≤3% CD19+ (Methods).
c
Mice with progressively growing tumors (sacrificed ≥225 mm2).
d
Mice were challenged with E.G7 lymphoma cells subcutaneously (1.0 x 106).
e
Total rejection rate of HSCT recipients was 90% (18/20).
Table 1 Figure. Kinetics of tumor growth and survival in previously vaccinated
HSCT recipients challenged with a lethal number of tumor cells. Previously
vaccinated transplant recipients (>100 d post-HSCT; Figures 4-6) were challenged with a
lethal number (1.0 x 106) of E.G7 tumor cells subcutaneously to determine if anti-tumor
memory had been generated by either vaccine strategy. (Left) Kinetics of tumor growth;
x: Untreated and Non-transplanted (pooled), ●: E.G7-gp96-Ig (individual), ■: E.G7gp96-Ig + IL-2S4B6 (individual). (Right) Survival post-challenge; n = 6-14 from pool of 2
subcutaneous challenge experiments; x: Untreated and Non-transplanted, ●: E.G7-gp96Ig, ■: E.G7-gp96-Ig + IL-2S4B6.
62
Figure 7A. IL-2 complex therapy enhanced NK cell numbers in HSCT recipients.
Transplants and treatments were performed as in Figure 5. IL-2S4B6 therapy elicited large
numbers of NK cells in the peritoneal cavity of intraperitoneally vaccinated HSCT
recipients 5 d following the 3rd treatment. NK1.1+ cell numbers in the peritoneal cavity;
n = 2.
63
Figure 7B. IL-2 complex therapy enhanced and anti-pathogen immunity in HSCT
recipients. Transplants were performed as in Figure 5 and HSCT recipients were infused
with IL-2S4B6 3 d post-HSCT and every 3 d for a total of 4 infusions. (Left) IL-2S4B6
induced expansion of CD8+ T cells in HSCT recipients immediately prior to Listeria
monocytogenes challenge. CD8+ T cell frequency in the peripheral blood 2 wk postHSCT; n = 5 from representative of 2 experiments; x: no HSCT, ●: HSCT, ■: HSCT +
IL-2S4B6. (Right) IL-2S4B6 treated HSCT recipients displayed fewer splenic bacterial CFU
following intravenous inoculation with 1.0 x 104 CFU Listeria monocytogenes 14 d postHSCT and 4 infusions of IL-2S4B6 starting 3 d post-HSCT and repeated every 3 d. CFU
numbers in the spleen 7 d post-infection; n = 5 from representative of 2 experiments; x:
no HSCT, ●: HSCT, ■: HSCT + IL-2S4B6.
64
Figure 8A: Effects of IL-2JES6. Transplants were performed as in Figure 5 and mice
received IL-2 pre-bound to anti-IL-2 mAb clone JES6 (IL-2JES6) 1 d following each
vaccination or in the absence of vaccination. (Left) IL-2JES6 only expanded CD8+ T cells
in combination with vaccination. CD8+ T cell frequency in the peripheral blood; n = 5; +:
IL-2JES6, ●: E.G7-gp96-Ig, ♦: E.G7-gp96-Ig + IL-2JES6. (Right) IL-2JES6 augmented
vaccine induced tumor-reactive CD8+ T cell expansion. Tumor-reactive CD8+ T cell
frequency in the peripheral blood; n = 5; +: IL-2JES6, ●: E.G7-gp96-Ig, ♦: E.G7-gp96-Ig +
IL-2JES6. (Bottom) Combination therapy with vaccine and IL-2JES6 enhanced MST and
overall survival; n = 5; +: IL-2JES6, ●: E.G7-gp96-Ig, ♦: E.G7-gp96-Ig + IL-2JES6.
65
Figure 8B. Comparison of IL-2mAb monotherapy. Composite data from Figures 5D and
8A. (Left) Monotherapy with IL-2JES6 did not expand CD8+ T cells as compared to IL2S4B6. CD8+ T cell frequency in the peripheral blood; n = 5-10 from pool of 2
experiments; ●: E.G7-gp96-Ig, x: IL-2S4B6, +: IL-2JES6. (Right) IL-2JES6 expanded tumorreactive CD8+ T cells in a subset of mice compared to minimal expansion by IL-2S4B6.
Tumor-reactive CD8+ T cell frequency in the peripheral blood; n = 5-10 from pool of 2
experiments; ●: E.G7-gp96-Ig, x: IL-2S4B6, +: IL-2JES6. (Bottom) Monotherapy with
either IL-2mAb reagent results in similar MST; n = 5-10 from pool of 2 experiments; ●:
E.G7-gp96-Ig, x: IL-2S4B6, +: IL-2JES6.
66
Figure 8C. Comparison of IL-2mAb in combination with vaccination. Composite data
from Figures 5C-D and 8A. (Left) Delayed expansion and increased contraction of CD8+
T cells following vaccination in combination with IL-2JES6 compared to IL-2S4B6. CD8+ T
cell frequency in the peripheral blood; n = 10 from pool of 2 experiments; ●: E.G7-gp96Ig, ■: E.G7-gp96-Ig + IL-2S4B6, ♦: E.G7-gp96-Ig + IL-2JES6. (Right) Augmented
expansion and contraction of tumor-reactive CD8+ T cells following vaccination in
combination with IL-2JES6 compared to IL-2S4B6. Tumor-reactive CD8+ T cell frequency
in the peripheral blood; n = 10 from pool of 2 experiments; ●: E.G7-gp96-Ig, ■: E.G7gp96-Ig + IL-2S4B6, ♦: E.G7-gp96-Ig + IL-2JES6. (Bottom) IL-2JES6 and IL-2S4B6 in
combination with vaccination enhanced MST and overall survival; n = 10 from pool of 2
experiments; ●: E.G7-gp96-Ig, ■: E.G7-gp96-Ig + IL-2S4B6, ♦: E.G7-gp96-Ig + IL-2JES6.
67
Table 2: Summary of IL-2/anti-IL-2 complex induced CD8+ T cell responses in the
presence and absence of vaccination with gp96-Ig secreting tumor cells.
Treatment
Donor CD8+ T cells
Tumor reactive CD8+ OT-I T cells
no vaccine
+/−a
IL-2S4B6
+
IL-2JES6
−
+b
gp96-Ig vaccine
IL-2S4B6
++
+
+
++
IL-2JES6
below level of detection
b
3/5
a
68
Figure 9. Depletion of NK cells in HSCT recipients resulted in diminished tumorreactive CD8+ T cell expansion and survival following vaccine and IL-2. Transplants,
treatments, and tumor inoculation were performed as in Figure 6. Anti-NK1.1 mAb clone
PK136 was administered 1 d before total body irradiation (200 μg) and 3 d (100 μg), 9 d
(50 μg), and 15 d (50 μg) post-HSCT. (Left) Depletion of NK cells in HSCT recipients
administered anti-NK1.1 mAb clone PK136 treated with vaccine and IL-2. Donor NK
cell frequency in the peripheral blood; n = 5; ■: E.G7-gp96-Ig + IL-2S4B6, □: E.G7-gp96Ig + IL-2S4B6 (αNK1.1 mAb). (Right) Depletion of NK cells in HSCT recipients resulted
in delayed and diminished tumor-reactive CD8+ T cell expansion following with vaccine
and IL-2. Donor CD8+ OVA257-264+ T cell frequency in the peripheral blood; n = 5; ■:
E.G7-gp96-Ig + IL-2S4B6, □: E.G7-gp96-Ig + IL-2S4B6 (αNK1.1 mAb). (Bottom)
Diminished survival of HSCT recipients treated with vaccine and IL-2 depleted of NK
cells; n = 5; x: IL-2S4B6, ■: E.G7-gp96-Ig + IL-2S4B6, □: E.G7-gp96-Ig + IL-2S4B6
(αNK1.1 mAb)
WORKS CITED
1.
Cancer facts & figures 2014. Atlanta: American Cancer Society, (2014).
2.
Jemal, A., Simard, E.P., Dorell, C., Noone, A.-M., Markowitz, L.E., Kohler, B.,
Eheman, C., Saraiya, M., Bandi, P., Saslow, D., Cronin, K.A., Watson, M.,
Schiffman, M., Henley, S.J., Schymura, M.J., Anderson, R.N., Yankey, D. &
Edwards, B.K. Annual report to the nation on the status of cancer, 1975–2009,
featuring the burden and trends in human papillomavirus (HPV)–associated cancers
and HPV vaccination coverage levels. J. Natl. Cancer Inst. 105, 175-201, (2013).
3.
Facts 2013. Leukemia & Lymphoma Society, (2013).
4.
Kuppers, R. Mechanisms of B-cell lymphoma pathogenesis. Nat. Rev. Cancer 5,
251-262, (2005).
5.
Aifantis, I., Raetz, E. & Buonamici, S. Molecular pathogenesis of T-cell leukaemia
and lymphoma. Nat. Rev. Immunol. 8, 380-390, (2008).
6.
Gilman, A. The initial clinical trial of nitrogen mustard. Am. J. Surg. 105, 574-578,
(1963).
7.
Farber, S., Diamond, L.K., Mercer, R.D., Sylvester, R.F. & Wolff, J.A. Temporary
remissions in acute leukemia in children produced by folic acid antagonist, 4aminopteroyl-glutamic acid (aminopterin). N. Engl. J. Med. 238, 787-793, (1948).
8.
Barrett, D.M., Singh, N., Porter, D.L., Grupp, S.A. & June, C.H. Chimeric antigen
receptor therapy for cancer. Annu. Rev. Med. 65, 333-347, (2014).
9.
Kalos, M., Levine, B.L., Porter, D.L., Katz, S., Grupp, S.A., Bagg, A. & June, C.H.
T cells with chimeric antigen receptors have potent antitumor effects and can
establish memory in patients with advanced leukemia. Sci. Trans. Med. 3, 95ra73,
(2011).
10.
Porter, D.L., Levine, B.L., Kalos, M., Bagg, A. & June, C.H. Chimeric antigen
receptor–modified T cells in chronic lymphoid leukemia. N. Engl. J. Med. 365,
725-733, (2011).
11.
Kochenderfer, J.N., Wilson, W.H., Janik, J.E., Dudley, M.E., Stetler-Stevenson, M.,
Feldman, S.A., Maric, I., Raffeld, M., Nathan, D.-A.N., Lanier, B.J., Morgan, R.A.
& Rosenberg, S.A. Eradication of B-lineage cells and regression of lymphoma in a
patient treated with autologous T cells genetically engineered to recognize CD19.
Blood 116, 4099-4102, (2010).
69
70
12.
Kochenderfer, J.N., Dudley, M.E., Feldman, S.A., Wilson, W.H., Spaner, D.E.,
Maric, I., Stetler-Stevenson, M., Phan, G.Q., Hughes, M.S., Sherry, R.M., Yang,
J.C., Kammula, U.S., Devillier, L., Carpenter, R., Nathan, D.-A.N., Morgan, R.A.,
Laurencot, C. & Rosenberg, S.A. B-cell depletion and remissions of malignancy
along with cytokine-associated toxicity in a clinical trial of anti-CD19 chimericantigen-receptor–transduced T cells. Blood 119, 2709-2720, (2012).
13.
Brentjens, R.J., Rivière, I., Park, J.H., Davila, M.L., Wang, X., Stefanski, J., Taylor,
C., Yeh, R., Bartido, S., Borquez-Ojeda, O., Olszewska, M., Bernal, Y., Pegram,
H., Przybylowski, M., Hollyman, D., Usachenko, Y., Pirraglia, D., Hosey, J.,
Santos, E., Halton, E., Maslak, P., Scheinberg, D., Jurcic, J., Heaney, M., Heller,
G., Frattini, M. & Sadelain, M. Safety and persistence of adoptively transferred
autologous CD19-targeted T cells in patients with relapsed or chemotherapy
refractory B-cell leukemias. Blood 118, 4817-4828, (2011).
14.
Savoldo, B., Ramos, C.A., Liu, E., Mims, M.P., Keating, M.J., Carrum, G.,
Kamble, R.T., Bollard, C.M., Gee, A.P., Mei, Z., Liu, H., Grilley, B., Rooney,
C.M., Heslop, H.E., Brenner, M.K. & Dotti, G. CD28 costimulation improves
expansion and persistence of chimeric antigen receptor–modified T cells in
lymphoma patients. J. Clin. Invest. 121, 1822-1826, (2011).
15.
Till, B.G., Jensen, M.C., Wang, J., Qian, X., Gopal, A.K., Maloney, D.G.,
Lindgren, C.G., Lin, Y., Pagel, J.M., Budde, L.E., Raubitschek, A., Forman, S.J.,
Greenberg, P.D., Riddell, S.R. & Press, O.W. CD20-specific adoptive
immunotherapy for lymphoma using a chimeric antigen receptor with both CD28
and 4-1BB domains: pilot clinical trial results. Blood 119, 3940-3950, (2012).
16.
Grupp, S.A., Kalos, M., Barrett, D., Aplenc, R., Porter, D.L., Rheingold, S.R.,
Teachey, D.T., Chew, A., Hauck, B., Wright, J.F., Milone, M.C., Levine, B.L. &
June, C.H. Chimeric antigen receptor–modified T cells for acute lymphoid
leukemia. N. Engl. J. Med. 368, 1509-1518, (2013).
17.
Attal, M., Harousseau, J.-L., Stoppa, A.-M., Sotto, J.-J., Fuzibet, J.-G., Rossi, J.-F.,
Casassus, P., Maisonneuve, H., Facon, T., Ifrah, N., Payen, C. & Bataille, R. A
prospective, randomized trial of autologous bone marrow transplantation and
chemotherapy in multiple myeloma. N. Engl. J. Med. 335, 91-97, (1996).
18.
Palumbo, A. & Anderson, K. Multiple myeloma. N. Engl. J. Med. 364, 1046-1060,
(2011).
19.
Jenq, R.R. & van den Brink, M.R. Allogeneic haematopoietic stem cell
transplantation: individualized stem cell and immune therapy of cancer. Nat. Rev.
Cancer 10, 213-221, (2010).
20.
Jacobson, L.O., Simmons, E.L., Marks, E.K., Robson, M.J., Bethard, W.F. &
Gaston, E.O. The role of the spleen in radiation injury and recovery. J. Lab. Clin.
Med. 35, 746-770, (1950).
71
21.
Lorenz, E., Uphoff, D., Reid, T.R. & Shelton, E. Modification of irradiation injury
in mice and guinea pigs by bone marrow injections. J. Natl. Cancer Inst. 12, 197201, (1951).
22.
Lindsley, D.L., Odell, T.T. & Tausche, F.G. Implantation of functional
erythropoietic elements following total-body irradiation. Proc. Soc. Exp. Biol. Med.
90, 512-515, (1955).
23.
Nowell, P.C., Cole, L.J., Habermeyer, J.G. & Roan, P.L. Growth and continued
function of rat marrow cells in X-radiated mice. Cancer Res. 16, 258-261, (1956).
24.
Ford, C.E., Hamerton, J.L., Barnes, D.W. & Loutit, J.F. Cytological identification
of radiation-chimaeras. Nature 177, 452-454, (1956).
25.
Mitchison, N.A. The colonisation of irradiated tissue by transplanted spleen cells.
Br. J. Exp. Pathol. 37, 239-247, (1956).
26.
Urso, P. & Congdon, C.C. The effect of the amount of isologous bone marrow
injected on the recovery of hematopoietic organs, survival and body weight after
lethal irradiation injury in mice. Blood 12, 251-260, (1957).
27.
Thomas, E.D., Lochte, H.L., Lu, W.C. & Ferrebee, J.W. Intravenous infusion of
bone marrow in patients receiving radiation and chemotherapy. N. Engl. J. Med.
257, 491-496, (1957).
28.
Pasquini, M. & Wang, Z. Current use and outcome of hematopoietic stem cell
transplantation: CIBMTR summary slides. CIBMTR, (2013).
29.
Berry, D.A., Ueno, N.T., Johnson, M.M., Lei, X., Caputo, J., Rodenhuis, S., Peters,
W.P., Leonard, R.C., Barlow, W.E., Tallman, M.S., Bergh, J., Nitz, U.A., Gianni,
A.M., Basser, R.L., Zander, A.R., Coombes, R.C., Roché, H., Tokuda, Y., de Vries,
E.G.E., Hortobagyi, G.N., Crown, J.P., Pedrazzoli, P., Bregni, M. & Demirer, T.
High-dose chemotherapy with autologous stem-cell support as adjuvant therapy in
breast cancer: overview of 15 randomized trials. J. Clin. Oncol. 29, 3214-3223,
(2011).
30.
Wang, J., Zhang, Q., Zhou, R., Chen, B. & Ouyang, J. High-dose chemotherapy
followed by autologous stem cell transplantation as a first-line therapy for high-risk
primary breast cancer: A meta-analysis. PLoS One 7, e33388, (2012).
31.
Kantoff, P.W., Higano, C.S., Shore, N.D., Berger, E.R., Small, E.J., Penson, D.F.,
Redfern, C.H., Ferrari, A.C., Dreicer, R., Sims, R.B., Xu, Y., Frohlich, M.W. &
Schellhammer, P.F. Sipuleucel-T immunotherapy for castration-resistant prostate
cancer. N. Engl. J. Med. 363, 411-422, (2010).
72
32.
Botrel, T.E., Clark, O., Pompeo, A.C., Bretas, F.F., Sadi, M.V., Ferreira, U. & Reis,
R.B. Immunotherapy with Sipuleucel-T (APC8015) in patients with metastatic
castration-refractory prostate cancer (mCRPC): a systematic review and metaanalysis. Int. Braz. J. Urol. 38, 717-727, (2012).
33.
Gross, L. Intradermal immunization of C3H mice against a sarcoma that originated
in an animal of the same line. Cancer Res. 3, 326-333, (1943).
34.
Foley, E.J. Antigenic properties of methylcholanthrene-induced tumors in mice of
the strain of origin. Cancer Res. 13, 835-837, (1953).
35.
Prehn, R.T. & Main, J.M. Immunity to methylcholanthrene-induced sarcomas. J.
Natl. Cancer Inst. 18, 769-778, (1957).
36.
Klein, G., Sjögren, H.O., Klein, E. & Hellström, K.E. Demonstration of resistance
against methylcholanthrene-induced sarcomas in the primary autochthonous host.
Cancer Res. 20, 1561-1572, (1960).
37.
Old, L.J., Boyse, E.A., Clarke, D.A. & Carswell, E.A. Antigenic properties of
chemically induced tumors. Ann. N. Y. Acad. Sci. 101, 80-106, (1962).
38.
Kaminski, M.S., Kitamura, K., Maloney, D.G. & Levy, R. Idiotype vaccination
against murine B cell lymphoma. Inhibition of tumor immunity by free idiotype
protein. J. Immunol. 138, 1289-1296, (1987).
39.
Kwak, L.W., Campbell, M.J., Czerwinski, D.K., Hart, S., Miller, R.A. & Levy, R.
Induction of immune responses in patients with B-cell lymphoma against the
surface-immunoglobulin idiotype expressed by their tumors. N. Engl. J. Med. 327,
1209-1215, (1992).
40.
Hsu, F.J., Kwak, L., Campbell, M., Liles, T., Czerwinski, D., Hart, S., Syrengelas,
A., Miller, R. & Levy, R. Clinical trials of idiotype-specific vaccine in B-cell
lymphomas. Ann. N. Y. Acad. Sci. 690, 385-387, (1993).
41.
Hsu, F.J., Caspar, C.B., Czerwinski, D., Kwak, L.W., Liles, T.M., Syrengelas, A.,
Taidi-Laskowski, B. & Levy, R. Tumor-specific idiotype vaccines in the treatment
of patients with B-cell lymphoma – Long-term results of a clinical trial. Blood 89,
3129-3135, (1997).
42.
Österborg, A., Yi, Q., Henriksson, L., Fagerberg, J., Bergenbrant, S., Jeddi-Tehrani,
M., Rudén, U., Lefvert, A.-K., Holm, G. & Mellstedt, H. Idiotype immunization
combined with granulocyte-macrophage colony-stimulating factor in myeloma
patients induced type I, major histocompatibility complex–restricted, CD8- and
CD4-specific T-cell responses. Blood 91, 2459-2466, (1998).
43.
Srivastava, P.K., DeLeo, A.B. & Old, L.J. Tumor rejection antigens of chemically
induced sarcomas of inbred mice. Proc. Natl. Acad. Sci. U. S. A. 83, 3407-3411,
(1986).
73
44.
Younes, A. A phase II study of heat shock protein-peptide complex-96 vaccine
therapy in patients with indolent non-Hodgkin's lymphoma. Clin. Lymphoma 4,
183-185, (2003).
45.
Oki, Y., McLaughlin, P., Fayad, L.E., Pro, B., Mansfield, P.F., Clayman, G.L.,
Medeiros, L.J., Kwak, L.W., Srivastava, P.K. & Younes, A. Experience with heat
shock protein-peptide complex 96 vaccine therapy in patients with indolent nonHodgkin lymphoma. Cancer 109, 77-83, (2007).
46.
Randazzo, M., Terness, P., Opelz, G. & Kleist, C. Active-specific immunotherapy
of human cancers with the heat shock protein gp96—revisited. Int. J. Cancer 130,
2219-2231, (2012).
47.
Yamazaki, K., Nguyen, T. & Podack, E.R. Cutting Edge: Tumor secreted heat
shock-fusion protein elicits CD8 cells for rejection. J. Immunol. 163, 5178-5182,
(1999).
48.
Strbo, N., Yamazaki, K., Lee, K., Rukavina, D. & Podack, E.R. Heat shock fusion
protein gp96-Ig mediates strong CD8 CTL expansion in vivo. Am. J. Reprod.
Immunol. 48, 220-225, (2002).
49.
Strbo, N., Oizumi, S., Sotosek-Tokmadzic, V. & Podack, E.R. Perforin is required
for innate and adaptive immunity induced by heat shock protein gp96. Immunity 18,
381-390, (2003).
50.
Oizumi, S., Strbo, N., Pahwa, S., Deyev, V. & Podack, E.R. Molecular and cellular
requirements for enhanced antigen cross-presentation to CD8 cytotoxic T
lymphocytes. J. Immunol. 179, 2310-2317, (2007).
51.
Oizumi, S., Deyev, V., Yamazaki, K., Schreiber, T., Strbo, N., Rosenblatt, J. &
Podack, E.R. Surmounting tumor-induced immune suppression by frequent
vaccination or immunization in the absence of B cells. J. Immunother. 31, 394-401,
(2008).
52.
Schreiber, T.H., Deyev, V.V., Rosenblatt, J.D. & Podack, E.R. Tumor-induced
suppression of CTL expansion and subjugation by gp96-Ig vaccination. Cancer
Res. 69, 2026-2033, (2009).
53.
Strbo, N., Pahwa, S., Kolber, M.A., Gonzalez, L., Fisher, E. & Podack, E.R. Cellsecreted gp96-Ig-peptide complexes induce lamina propria and intraepithelial CD8+
cytotoxic T lymphocytes in the intestinal mucosa. Mucosal Immunol. 3, 182-192,
(2010).
54.
Strbo, N., Vaccari, M., Pahwa, S., Kolber, M.A., Fisher, E., Gonzalez, L., Doster,
M.N., Hryniewicz, A., Felber, B.K., Pavlakis, G.N., Franchini, G. & Podack, E.R.
Gp96 SIV Ig immunization induces potent polyepitope specific, multifunctional
memory responses in rectal and vaginal mucosa. Vaccine 29, 2619-2625, (2011).
74
55.
Strbo, N., Vaccari, M., Pahwa, S., Kolber, M.A., Doster, M.N., Fisher, E.,
Gonzalez, L., Stablein, D., Franchini, G. & Podack, E.R. Cutting Edge: Novel
vaccination modality provides significant protection against mucosal infection by
highly pathogenic simian immunodeficiency virus. J. Immunol. 190, 2495-2499,
(2013).
56.
Raez, L.E., Walker, G.R., Baldie, P., Fisher, E., Gomez, J.E., Tolba, K., Santos,
E.S. & Podack, E.R. CD8 T cell response in a phase I study of therapeutic
vaccination of advanced NSCLC with allogeneic tumor cells secreting endoplasmic
reticulum-chaperone gp96-Ig-peptide complexes. Advances in Lung Cancer 2, 918, (2013).
57.
Morgan, D.A., Ruscetti, F.W. & Gallo, R. Selective in vitro growth of T
lymphocytes from normal human bone marrows. Science 193, 1007-1008, (1976).
58.
Taniguchi, T., Matsui, H., Fujita, T., Takaoka, C., Kashima, N., Yoshimoto, R. &
Hamuro, J. Structure and expression of a cloned cDNA for human interleukin-2.
Nature 302, 305-310, (1983).
59.
Harada, M., Matsuzaki, G., Shinomiya, Y., Kurosawa, S., Ito, O., Okamoto, T.,
Takenoyama, M., Sumitika, H., Nishimura, Y. & Nomoto, K. Generation of tumorspecific cytotoxic T lymphocytes in vivo by combined treatment with inactivated
tumor cells and recombinant interleukin-2. Cancer Immunol. Immunother. 38, 332338, (1994).
60.
Cao, X., Zhang, W., Gu, S., Yu, Y., Tao, Q. & Ye, T. Induction of antitumor
immunity and treatment of preestablished tumor by interleukin-6-gene-transfected
melanoma cells combined with low-dose interleukin-2. J. Cancer Res. Clin. Oncol.
121, 721-728, (1995).
61.
Shimizu, K., Fields, R.C., Giedlin, M. & Mulé, J.J. Systemic administration of
interleukin 2 enhances the therapeutic efficacy of dendritic cell-based tumor
vaccines. Proc. Natl. Acad. Sci. U. S. A. 96, 2268-2273, (1999).
62.
Schrayer, D., Kouttab, N., Hearing, V. & Wanebo, H. Synergistic effect of
interleukin-2 and a vaccine of irradiated melanoma cells transfected to secrete
staphylococcal enterotoxin A. Clin. Exp. Metastasis 19, 43-53, (2002).
63.
Kochenderfer, J.N., Chien, C.D., Simpson, J.L. & Gress, R.E. Synergism between
CpG-containing oligodeoxynucleotides and IL-2 causes dramatic enhancement of
vaccine-elicited CD8+ T cell responses. J. Immunol. 177, 8860-8873, (2006).
64.
Kochenderfer, J.N., Chien, C.D., Simpson, J.L. & Gress, R.E. Maximizing CD8+ T
cell responses elicited by peptide vaccines containing CpG oligodeoxynucleotides.
Clin. Immunol. 124, 119-130, (2007).
75
65.
Kochenderfer, J.N., Simpson, J.L., Chien, C.D. & Gress, R.E. Vaccination
regimens incorporating CpG-containing oligodeoxynucleotides and IL-2 generate
antigen-specific antitumor immunity from T-cell populations undergoing
homeostatic peripheral expansion after BMT. Blood 110, 450-460, (2007).
66.
Sato, J., Hamaguchi, N., Doken, K., Gotoh, K., Ootsu, K., Iwasa, S., Ogawa, Y. &
Toguchi, H. Enhancement of anti-tumor activity of recombinant interleukin-2 (rIL2) by immunocomplexing with a monoclonal antibody against rIL-2. Biotherapy 6,
225-231, (1993).
67.
Courtney, L.P., Phelps, J.L. & Karavodin, L.M. An anti-IL-2 antibody increases
serum halflife and improves anti-tumor efficacy of human recombinant interleukin2. Immunopharmacol. 28, 223-232, (1994).
68.
Phelan, J.D., Orekov, T. & Finkelman, F.D. Cutting Edge: Mechanism of
enhancement of in vivo cytokine effects by anti-cytokine monoclonal antibodies. J.
Immunol. 180, 44-48, (2008).
69.
Tomala, J., Chmelova, H., Mrkvan, T., Rihova, B. & Kovar, M. In vivo expansion
of activated naive CD8+ T cells and NK cells driven by complexes of IL-2 and antiIL-2 monoclonal antibody as novel approach of cancer immunotherapy. J.
Immunol. 183, 4904-4912, (2009).
70.
Létourneau, S., van Leeuwen, E.M.M., Krieg, C., Martin, C., Pantaleo, G., Sprent,
J., Surh, C.D. & Boyman, O. IL-2/anti-IL-2 antibody complexes show strong
biological activity by avoiding interaction with IL-2 receptor α subunit CD25. Proc.
Natl. Acad. Sci. U. S. A. 107, 2171-2176, (2010).
71.
Boyman, O., Kovar, M., Rubinstein, M.P., Surh, C.D. & Sprent, J. Selective
stimulation of T cell subsets with antibody-cytokine immune complexes. Science
311, 1924-1927, (2006).
72.
Murali-Krishna, K. & Ahmed, R. Cutting Edge: Naive T cells masquerading as
memory cells. J. Immunol. 165, 1733-1737, (2000).
73.
Goldrath, A.W., Bogatzki, L.Y. & Bevan, M.J. Naive T cells transiently acquire a
memory-like phenotype during homeostasis-driven proliferation. J. Exp. Med. 192,
557-564, (2000).
74.
Clarkson, B., Thompson, D., Horwith, M. & Luckey, E.H. Cyclical edema and
shock due to increased capillary permeability. Am. J. Med. 29, 193-216, (1960).
75.
Krieg, C., Létourneau, S., Pantaleo, G. & Boyman, O. Improved IL-2
immunotherapy by selective stimulation of IL-2 receptors on lymphocytes and
endothelial cells. Proc. Natl. Acad. Sci. U. S. A. 107, 11906-11911, (2010).
76
76.
Mostböck, S., Lutsiak, M.E.C., Milenic, D.E., Baidoo, K., Schlom, J. & Sabzevari,
H. IL-2/anti-IL-2 antibody complex enhances vaccine-mediated antigen-specific
CD8+ T cell responses and increases the ratio of effector/memory CD8+ T cells to
regulatory T cells. J. Immunol. 180, 5118-5129, (2008).
77.
Jin, G.-H., Hirano, T. & Murakami, M. Combination treatment with IL-2 and antiIL-2 mAbs reduces tumor metastasis via NK cell activation. Int. Immunol. 20, 783789, (2008).
78.
Levin, A.M., Bates, D.L., Ring, A.M., Krieg, C., Lin, J.T., Su, L., Moraga, I.,
Raeber, M.E., Bowman, G.R., Novick, P., Pande, V.S., Fathman, C.G., Boyman, O.
& Garcia, K.C. Exploiting a natural conformational switch to engineer an
interleukin-2 'superkine'. Nature 484, 529-533, (2012).
79.
Pope, C., Kim, S.-K., Marzo, A., Williams, K., Jiang, J., Shen, H. & Lefrançois, L.
Organ-specific regulation of the CD8 T cell response to Listeria monocytogenes
infection. J. Immunol. 166, 3402-3409, (2001).
80.
Hamilton, S.E., Schenkel, J.M., Akue, A.D. & Jameson, S.C. IL-2 complex
treatment can protect naive mice from bacterial and viral infection. J. Immunol.
185, 6584-6590, (2010).
81.
Castro, I., Dee, M.J. & Malek, T.R. Transient enhanced IL-2R signaling early
during priming rapidly amplifies development of functional CD8+ T effectormemory cells. J. Immunol. 189, 4321-4330, (2012).
82.
Kamimura, D. & Bevan, M.J. Naive CD8+ T cells differentiate into protective
memory-like cells after IL-2–anti–IL-2 complex treatment in vivo. J. Exp. Med.
204, 1803-1812, (2007).
83.
Higuchi, C., Thompson, J., Petersen, F., Buckner, C. & Fefer, A. Toxicity and
immunomodulatory effects of interleukin-2 after autologous bone marrow
transplantation for hematologic malignancies. Blood 77, 2561-2568, (1991).
84.
Benyunes, M.C., Massumoto, C., York, A., Higuchi, C.M., Buckner, C.D.,
Thompson, J.A., Petersen, F.B. & Fefer, A. Interleukin-2 with or without
lymphokine-activated killer cells as consolidative immunotherapy after autologous
bone marrow transplantation for acute myelogenous leukemia. Bone Marrow
Transplant. 12, 159-163, (1993).
85.
Benyunes, M.C., Higuchi, C., York, A., Lindgren, C., Thompson, J.A., Buckner,
C.D. & Fefer, A. Immunotherapy with interleukin 2 with or without lymphokineactivated killer cells after autologous bone marrow transplantation for malignant
lymphoma: a feasibility trial. Bone Marrow Transplant. 16, 283-288, (1995).
77
86.
Weisdorf, D.J., Anderson, P.M., Blazar, B.R., Uckun, F.M., Kersey, J.H. &
Ramsay, N.K. Interleukin 2 immediately after autologous bone marrow
transplantation for acute lymphoblastic leukemia--a phase I study. Transplantation
55, 61-66, (1993).
87.
Miller, J.S., Tessmer-Tuck, J., Pierson, B.A., Weisdorf, D., McGlave, P., Blazar,
B.R., Katsanis, E., Verfaillie, C., Lebkowski, J., Radford, J., Jr. & Burns, L.J. Low
dose subcutaneous interleukin-2 after autologous transplantation generates
sustained in vivo natural killer cell activity. Biol. Blood Marrow Transplant. 3, 3444, (1997).
88.
Attal, M., Blaise, D., Marit, G., Payen, C., Michallet, M., Vernant, J., Sauvage, C.,
Troussard, X., Nedellec, G. & Pico, J. Consolidation treatment of adult acute
lymphoblastic leukemia: a prospective, randomized trial comparing allogeneic
versus autologous bone marrow transplantation and testing the impact of
recombinant interleukin-2 after autologous bone marrow transplantation. BGMT
Group. Blood 86, 1619-1628, (1995).
89.
Blaise, D., Attal, M., Reiffers, J., Michallet, M., Bellanger, C., Pico, J.L., Stoppa,
A.M., Payen, C., Marit, G., Bouabdallah, R., Sotto, J.J., Rossi, J.F., Brandely, M.,
Hercend, T. & Maraninchi, D. Randomized study of recombinant interleukin-2 after
autologous bone marrow transplantation for acute leukemia in first complete
remission. Eur. Cytokine Netw. 11, 91-98, (2000).
90.
Robinson, N., Benyunes, M.C., Thompson, J.A., York, A., Petersdorf, S., Press, O.,
Lindgren, C., Chauncey, T., Buckner, C.D., Bensinger, W.I., Appelbaum, F.R. &
Fefer, A. Interleukin-2 after autologous stem cell transplantation for hematologic
malignancy: a phase I/II study. Bone Marrow Transplant. 19, 435-442, (1997).
91.
Burns, L.J., Weisdorf, D.J., DeFor, T.E., Vesole, D.H., Repka, T.L., Blazar, B.R.,
Burger, S.R., Panoskaltsis-Mortari, A., Keever-Taylor, C.A., Zhang, M.J. & Miller,
J.S. IL-2-based immunotherapy after autologous transplantation for lymphoma and
breast cancer induces immune activation and cytokine release: a phase I/II trial.
Bone Marrow Transplant. 32, 177-186, (2003).
92.
Thompson, J.A., Fisher, R.I., LeBlanc, M., Forman, S.J., Press, O.W., Unger, J.M.,
Nademanee, A.P., Stiff, P.J., Petersdorf, S.H. & Fefer, A. Total body irradiation,
etoposide, cyclophosphamide, and autologous peripheral blood stem-cell
transplantation followed by randomization to therapy with interleukin-2 versus
observation for patients with non-Hodgkin lymphoma: results of a phase 3
randomized trial by the Southwest Oncology Group (SWOG 9438). Blood 111,
4048-4054, (2008).
78
93.
Buyse, M., Squifflet, P., Lange, B.J., Alonzo, T.A., Larson, R.A., Kolitz, J.E.,
George, S.L., Bloomfield, C.D., Castaigne, S., Chevret, S., Blaise, D., Maraninchi,
D., Lucchesi, K.J. & Burzykowski, T. Individual patient data meta-analysis of
randomized trials evaluating IL-2 monotherapy as remission maintenance therapy
in acute myeloid leukemia. Blood 117, 7007-7013, (2011).
94.
Mackall, C., Bare, C., Granger, L., Sharrow, S., Titus, J. & Gress, R. Thymicindependent T cell regeneration occurs via antigen-driven expansion of peripheral T
cells resulting in a repertoire that is limited in diversity and prone to skewing. J.
Immunol. 156, 4609-4616, (1996).
95.
Dummer, W., Niethammer, A.G., Baccala, R., Lawson, B.R., Wagner, N., Reisfeld,
R.A. & Theofilopoulos, A.N. T cell homeostatic proliferation elicits effective
antitumor autoimmunity. J. Clin. Invest. 110, 185-192, (2002).
96.
Hu, H.-M., Poehlein, C.H., Urba, W.J. & Fox, B.A. Development of antitumor
immune responses in reconstituted lymphopenic hosts. Cancer Res. 62, 3914-3919,
(2002).
97.
Ma, J., Urba, W.J., Si, L., Wang, Y., Fox, B.A. & Hu, H.-M. Anti-tumor T cell
response and protective immunity in mice that received sublethal irradiation and
immune reconstitution. Eur. J. Immunol. 33, 2123-2132, (2003).
98.
Wang, L.-X., Li, R., Yang, G., Lim, M., O'Hara, A., Chu, Y., Fox, B.A., Restifo,
N.P., Urba, W.J. & Hu, H.-M. Interleukin-7-dependent expansion and persistence
of melanoma-specific T cells in lymphodepleted mice lead to tumor regression and
editing. Cancer Res. 65, 10569-10577, (2005).
99.
Keith, M.R. & Levy, R.B. Transplant conditions determine the contribution of
homeostatically expanded donor CD8 memory cells to host lymphoid reconstitution
following syngeneic HCT. Exp. Hematol. 35, 1303-1315, (2007).
100. Wrzesinski, C., Paulos, C.M., Kaiser, A., Muranski, P., Palmer, D.C., Gattinoni, L.,
Yu, Z., Rosenberg, S.A. & Restifo, N.P. Increased intensity lymphodepletion
enhances tumor treatment efficacy of adoptively transferred tumor-specific T cells.
J. Immunother. 33, 1-7, (2010).
101. Udono, H., Levey, D.L. & Srivastava, P.K. Cellular requirements for tumorspecific immunity elicited by heat shock proteins: tumor rejection antigen gp96
primes CD8+ T cells in vivo. Proc. Natl. Acad. Sci. U. S. A. 91, 3077-3081, (1994).
102. Suto, R. & Srivastava, P.K. A mechanism for the specific immunogenicity of heat
shock protein-chaperoned peptides. Science 269, 1585-1588, (1995).
103. Tamura, Y., Peng, P., Liu, K., Daou, M. & Srivastava, P.K. Immunotherapy of
tumors with autologous tumor-derived heat shock protein preparations. Science
278, 117-120, (1997).
79
104. Janetzki, S., Blachere, N.E. & Srivastava, P.K. Generation of tumor-specific
cytotoxic T lymphocytes and memory T cells by immunization with tumor-derived
heat shock protein gp96. J. Immunother. 21, 269-276, (1998).
105. Binder, R.J. & Srivastava, P.K. Peptides chaperoned by heat-shock proteins are a
necessary and sufficient source of antigen in the cross-priming of CD8+ T cells.
Nat. Immunol. 6, 593-599, (2005).
106. Binder, R.J., Kelly, J.B., Vatner, R.E. & Srivastava, P.K. Specific immunogenicity
of heat shock protein gp96 derives from chaperoned antigenic peptides and not
from contaminating proteins. J. Immunol. 179, 7254-7261, (2007).
107. Basu, S., Binder, R.J., Suto, R., Anderson, K.M. & Srivastava, P.K. Necrotic but
not apoptotic cell death releases heat shock proteins, which deliver a partial
maturation signal to dendritic cells and activate the NF-κB pathway. Int. Immunol.
12, 1539-1546, (2000).
108. Vabulas, R.M., Braedel, S., Hilf, N., Singh-Jasuja, H., Herter, S., Ahmad-Nejad, P.,
Kirschning, C.J., da Costa, C., Rammensee, H.-G., Wagner, H. & Schild, H. The
endoplasmic reticulum-resident heat shock protein gp96 activates dendritic cells via
the toll-like receptor 2/4 pathway. J. Biol. Chem. 277, 20847-20853, (2002).
109. Binder, R.J., Han, D.K. & Srivastava, P.K. CD91: a receptor for heat shock protein
gp96. Nat. Immunol. 1, 151-155, (2000).
110. Matzinger, P. & Bevan, M.J. Induction of H-2-restricted cytotoxic T cells: in vivo
induction has the appearance of being unrestricted. Cell. Immunol. 33, 92-100,
(1977).
111. Arnold, D., Faath, S., Rammensee, H. & Schild, H. Cross-priming of minor
histocompatibility antigen-specific cytotoxic T cells upon immunization with the
heat shock protein gp96. J. Exp. Med. 182, 885-889, (1995).
112. Slavin, S., Ackerstein, A., Kedar, E. & Weiss, L. IL-2 activated cell-mediated
immunotherapy: control of minimal residual disease in malignant disorders by
allogeneic lymphocytes and IL-2. Bone Marrow Transplant. 6 Suppl 1, 86-90,
(1990).
113. Ackerstein, A., Kedar, E. & Slavin, S. Use of recombinant human interleukin-2 in
conjunction with syngeneic bone marrow transplantation in mice as a model for
control of minimal residual disease in malignant hematologic disorders. Blood 78,
1212-1215, (1991).
114. Piccirillo, C.A. & Shevach, E.M. Cutting Edge: Control of CD8+ T cell activation
by CD4+CD25+ immunoregulatory cells. J. Immunol. 167, 1137-1140, (2001).
80
115. Borriello, F., Sethna, M.P., Boyd, S.D., Schweitzer, A.N., Tivol, E.A., Jacoby, D.,
Strom, T.B., Simpson, E.M., Freeman, G.J. & Sharpe, A.H. B7-1 and B7-2 have
overlapping, critical roles in immunoglobulin class switching and germinal center
formation. Immunity 6, 303-313, (1997).
116. Hogquist, K.A., Jameson, S.C., Heath, W.R., Howard, J.L., Bevan, M.J. &
Carbone, F.R. T cell receptor antagonist peptides induce positive selection. Cell 76,
17-27, (1994).
117. Mombaerts, P., Iacomini, J., Johnson, R.S., Herrup, K., Tonegawa, S. &
Papaioannou, V.E. RAG-1-deficient mice have no mature B and T lymphocytes.
Cell 68, 869-877, (1992).
118. Hildner, K., Edelson, B.T., Purtha, W.E., Diamond, M., Matsushita, H., Kohyama,
M., Calderon, B., Schraml, B.U., Unanue, E.R., Diamond, M.S., Schreiber, R.D.,
Murphy, T.L. & Murphy, K.M. Batf3 deficiency reveals a critical role for CD8α+
dendritic cells in cytotoxic T cell immunity. Science 322, 1097-1100, (2008).
119. Toubai, T., Sun, Y., Luker, G., Liu, J., Luker, K.E., Tawara, I., Evers, R., Liu, C.,
Mathewson, N., Malter, C., Nieves, E., Choi, S., Murphy, K.M. & Reddy, P. Hostderived CD8+ dendritic cells are required for induction of optimal graft-versustumor responses after experimental allogeneic bone marrow transplantation. Blood
121, 4231-4241, (2013).
120. Kagi, D., Ledermann, B., Burki, K., Seiler, P., Odermatt, B., Olsen, K.J., Podack,
E.R., Zinkernagel, R.M. & Hengartner, H. Cytotoxicity mediated by T cells and
natural killer cells is greatly impaired in perforin-deficient mice. Nature 369, 31-37,
(1994).
121. Wan, Y.Y. & Flavell, R.A. Identifying Foxp3-expressing suppressor T cells with a
bicistronic reporter. Proc. Natl. Acad. Sci. U. S. A. 102, 5126-5131, (2005).
122. Gorer, P.A. Studies in antibody response of mice to tumour inoculation. Br. J.
Cancer 4, 372-379, (1950).
123. Moore, M.W., Carbone, F.R. & Bevan, M.J. Introduction of soluble protein into the
class I pathway of antigen processing and presentation. Cell 54, 777-785, (1988).
124. Urbieta, M., Barao, I., Jones, M., Jurecic, R., Panoskaltsis-Mortari, A., Blazar,
B.R., Murphy, W.J. & Levy, R.B. Hematopoietic progenitor cell regulation by
CD4+CD25+ T cells. Blood 115, 4934-4943, (2010).
125. Ross, D., Jones, M., Komanduri, K. & Levy, R.B. Antigen and lymphopenia-driven
donor T cells are differentially diminished by post-transplantation administration of
cyclophosphamide after hematopoietic cell transplantation. Biol. Blood Marrow
Transplant. 19, 1430-1438, (2013).
81
126. Foulds, K.E., Zenewicz, L.A., Shedlock, D.J., Jiang, J., Troy, A.E. & Shen, H.
Cutting Edge: CD4 and CD8 T cells are intrinsically different in their proliferative
responses. J. Immunol. 168, 1528-1532, (2002).
127. Geissmann, F., Jung, S. & Littman, D.R. Blood monocytes consist of two principal
subsets with distinct migratory properties. Immunity 19, 71-82, (2003).
128. Geissmann, F., Manz, M.G., Jung, S., Sieweke, M.H., Merad, M. & Ley, K.
Development of monocytes, macrophages, and dendritic cells. Science 327, 656661, (2010).
129. Blazar, B.R., Lees, C.J., Martin, P.J., Noelle, R.J., Kwon, B., Murphy, W. &
Taylor, P.A. Host T cells resist graft-versus-host disease mediated by donor
leukocyte infusions. J. Immunol. 165, 4901-4909, (2000).
130. Ernst, B., Lee, D.-S., Chang, J.M., Sprent, J. & Surh, C.D. The peptide ligands
mediating positive selection in the thymus control T cell survival and homeostatic
proliferation in the periphery. Immunity 11, 173-181, (1999).
131. Goldrath, A.W. & Bevan, M.J. Low-affinity ligands for the TCR drive proliferation
of mature CD8+ T cells in lymphopenic hosts. Immunity 11, 183-190, (1999).
132. Kieper, W.C. & Jameson, S.C. Homeostatic expansion and phenotypic conversion
of naïve T cells in response to self peptide/MHC ligands. Proc. Natl. Acad. Sci. U.
S. A. 96, 13306-13311, (1999).
133. Precopio, M.L., Betts, M.R., Parrino, J., Price, D.A., Gostick, E., Ambrozak, D.R.,
Asher, T.E., Douek, D.C., Harari, A., Pantaleo, G., Bailer, R., Graham, B.S.,
Roederer, M. & Koup, R.A. Immunization with vaccinia virus induces
polyfunctional and phenotypically distinctive CD8+ T cell responses. J. Exp. Med.
204, 1405-1416, (2007).
134. Korngold, R., Leighton, C. & Manser, T. Graft-versus-myeloid leukemia responses
following syngeneic and allogeneic bone marrow transplantation. Transplantation
58, 278-287, (1994).
135. Nguyen, C.L., Salem, M.L., Rubinstein, M.P., Demcheva, M., Vournakis, J.N.,
Cole, D.J. & Gillanders, W.E. Mechanisms of enhanced antigen-specific T cell
response following vaccination with a novel peptide-based cancer vaccine and
systemic interleukin-2 (IL-2). Vaccine 21, 2318-2328, (2003).
136. Blazar, B.R., Taylor, P.A., Boyer, M.W., Panoskaltsis-Mortari, A., Allison, J.P. &
Vallera, D.A. CD28/B7 interactions are required for sustaining the graft-versusleukemia effect of delayed post-bone marrow transplantation splenocyte infusion in
murine recipients of myeloid or lymphoid leukemia cells. J. Immunol. 159, 34603473, (1997).
82
137. Zhang, Y., Joe, G., Zhu, J., Carroll, R., Levine, B., Hexner, E., June, C. & Emerson,
S.G. Dendritic cell–activated CD44hiCD8+ T cells are defective in mediating acute
graft-versus-host disease but retain graft-versus-leukemia activity. Blood 103,
3970-3978, (2004).
138. Prlic, M., Kamimura, D. & Bevan, M.J. Rapid generation of a functional NK-cell
compartment. Blood 110, 2024-2026, (2007).
139. Rapoport, A.P., Aqui, N.A., Stadtmauer, E.A., Vogl, D.T., Fang, H.-B., Cai, L.,
Janofsky, S., Chew, A., Storek, J., Akpek, G., Badros, A., Yanovich, S., Tan, M.T.,
Veloso, E., Pasetti, M.F., Cross, A., Philip, S., Murphy, H., Bhagat, R., Zheng, Z.,
Milliron, T., Cotte, J., Cannon, A., Levine, B.L., Vonderheide, R.H. & June, C.H.
Combination immunotherapy using adoptive T-cell transfer and tumor antigen
vaccination on the basis of hTERT and survivin after ASCT for myeloma. Blood
117, 788-797, (2011).
140. Schreiber, T.H., Raez, L., Rosenblatt, J.D. & Podack, E.R. Tumor immunogenicity
and responsiveness to cancer vaccine therapy: The state of the art. Semin. Immunol.
22, 105-112, (2010).
141. Rapoport, A.P., Stadtmauer, E.A., Aqui, N., Badros, A., Cotte, J., Chrisley, L.,
Veloso, E., Zheng, Z., Westphal, S., Mair, R., Chi, N., Ratterree, B., Pochran, M.F.,
Natt, S., Hinkle, J., Sickles, C., Sohal, A., Ruehle, K., Lynch, C., Zhang, L., Porter,
D.L., Luger, S., Guo, C., Fang, H.-B., Blackwelder, W., Hankey, K., Mann, D.,
Edelman, R., Frasch, C., Levine, B.L., Cross, A. & June, C.H. Restoration of
immunity in lymphopenic individuals with cancer by vaccination and adoptive Tcell transfer. Nat. Med. 11, 1230-1237, (2005).
142. Stadtmauer, E.A., Vogl, D.T., Luning Prak, E., Boyer, J., Aqui, N.A., Rapoport,
A.P., McDonald, K.R., Hou, X., Murphy, H., Bhagat, R., Mangan, P.A., Chew, A.,
Veloso, E.A., Levine, B.L., Vonderheide, R.H., Jawad, A.F., June, C.H. & Sullivan,
K.E. Transfer of influenza vaccine–primed costimulated autologous T cells after
stem cell transplantation for multiple myeloma leads to reconstitution of influenza
immunity: results of a randomized clinical trial. Blood 117, 63-71, (2011).
143. Borrello, I., Sotomayor, E.M., Rattis, F.-M., Cooke, S.K., Gu, L. & Levitsky, H.I.
Sustaining the graft-versus-tumor effect through posttransplant immunization with
granulocyte-macrophage colony-stimulating factor (GM-CSF)–producing tumor
vaccines. Blood 95, 3011-3019, (2000).
144. Jing, W., Gershan, J.A. & Johnson, B.D. Depletion of CD4 T cells enhances
immunotherapy for neuroblastoma after syngeneic HSCT but compromises
development of antitumor immune memory. Blood 113, 4449-4457, (2009).
145. Jing, W., Yan, X., Hallett, W.H.D., Gershan, J.A. & Johnson, B.D. Depletion of
CD25+ T cells from hematopoietic stem cell grafts increases posttransplantation
vaccine-induced immunity to neuroblastoma. Blood 117, 6952-6962, (2011).
83
146. Hirokawa, K., Sado, T., Kubo, S., Kamisaku, H., Hitomi, K. & Utsuyama, M.
Intrathymic T cell differentiation in radiation bone marrow chimeras and its role in
T cell emigration to the spleen. An immunohistochemical study. J. Immunol. 134,
3615-3624, (1985).
147. Qian, J., Wang, S., Yang, J., Xie, J., Lin, P., Freeman, M.E. & Yi, Q. Targeting heat
shock proteins for immunotherapy in multiple myeloma: generation of myelomaspecific CTLs using dendritic cells pulsed with tumor-derived gp96. Clin. Cancer
Res. 11, 8808-8815, (2005).
148. Qian, J., Hong, S., Wang, S., Zhang, L., Sun, L., Wang, M., Yang, J., Kwak, L.W.,
Hou, J. & Yi, Q. Myeloma cell line-derived, pooled heat shock proteins as a
universal vaccine for immunotherapy of multiple myeloma. Blood 114, 3880-3889,
(2009).
149. Sato, K., Torimoto, Y., Tamura, Y., Shindo, M., Shinzaki, H., Hirai, K. & Kohgo,
Y. Immunotherapy using heat-shock protein preparations of leukemia cells after
syngeneic bone marrow transplantation in mice. Blood 98, 1852-1857, (2001).
150. Iuchi, Y., Torimoto, Y., Sato, K., Tamura, Y., Jimbo, J., Inamura, J., Shindo, M.,
Ikuta, K., Ohnishi, K. & Kohgo, Y. Combined use of dendritic cells enhances
specific antileukemia immunity by leukemia cell-derived heat shock protein 70 in a
mouse model with minimal residual leukemia cells. Int. J. Hematol. 84, 449-458,
(2006).
151. Jimbo, J., Sato, K., Hosoki, T., Shindo, M., Ikuta, K., Torimoto, Y. & Kohgo, Y.
Induction of leukemia-specific antibodies by immunotherapy with leukemia-cellderived heat shock protein 70. Cancer Sci. 99, 1427-1434, (2008).
152. Gerosa, F., Baldani-Guerra, B., Nisii, C., Marchesini, V., Carra, G. & Trinchieri, G.
Reciprocal activating interaction between natural killer cells and dendritic cells. J.
Exp. Med. 195, 327-333, (2002).
153. Piccioli, D., Sbrana, S., Melandri, E. & Valiante, N.M. Contact-dependent
stimulation and inhibition of dendritic cells by natural killer cells. J. Exp. Med. 195,
335-341, (2002).
154. Ferlazzo, G., Tsang, M.L., Moretta, L., Melioli, G., Steinman, R.M. & Münz, C.
Human dendritic cells activate resting natural killer (NK) cells and are recognized
via the NKp30 receptor by activated NK cells. J. Exp. Med. 195, 343-351, (2002).
155. Rubinstein, M.P., Kovar, M., Purton, J.F., Cho, J.-H., Boyman, O., Surh, C.D. &
Sprent, J. Converting IL-15 to a superagonist by binding to soluble IL-15Rα. Proc.
Natl. Acad. Sci. U. S. A. 103, 9166-9171, (2006).
156. Stoklasek, T.A., Schluns, K.S. & Lefrançois, L. Combined IL-15/IL-15Rα
immunotherapy maximizes IL-15 activity in vivo. J. Immunol. 177, 6072-6080,
(2006).
84
157. Mielke, M.E., Niedobitek, G., Stein, H. & Hahn, H. Acquired resistance to Listeria
monocytogenes is mediated by Lyt-2+ T cells independently of the influx of
monocytes into granulomatous lesions. J. Exp. Med. 170, 589-594, (1989).
158. Dunn, P.L. & North, R.J. Early gamma interferon production by natural killer cells
is important in defense against murine listeriosis. Infect. Immun. 59, 2892-2900,
(1991).
159. Blattman, J.N., Grayson, J.M., Wherry, E.J., Kaech, S.M., Smith, K.A. & Ahmed,
R. Therapeutic use of IL-2 to enhance antiviral T-cell responses in vivo. Nat. Med.
9, 540-547, (2003).
160. Molloy, M.J., Zhang, W. & Usherwood, E.J. Cutting Edge: IL-2 immune
complexes as a therapy for persistent virus infection. J. Immunol. 182, 4512-4515,
(2009).
161. Shatry, A. & Levy, R.B. In situ activation and expansion of host tregs: a new
approach to enhance donor chimerism and stable engraftment in major
histocompatibility complex-matched allogeneic hematopoietic cell transplantation.
Biol. Blood Marrow Transplant. 15, 785-794, (2009).
162. Kennedy, M.K., Glaccum, M., Brown, S.N., Butz, E.A., Viney, J.L., Embers, M.,
Matsuki, N., Charrier, K., Sedger, L., Willis, C.R., Brasel, K., Morrissey, P.J.,
Stocking, K., Schuh, J.C.L., Joyce, S. & Peschon, J.J. Reversible defects in natural
killer and memory CD8 T cell lineages in interleukin 15–deficient mice. J. Exp.
Med. 191, 771-780, (2000).