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Prepublished online as a Blood First Edition Paper on August 22, 2002; DOI 10.1182/blood-2002-05-1580.
IMMUNOBIOLOGY
From the Department of Pediatrics, Steele Memorial
Children's Research Center, University of Arizona, Tucson.
We have previously reported that apoptotic tumor cells can be
either immunogenic or nonimmunogenic in vivo, depending on whether or
not these cells are heat stressed before induction of apoptosis. Stressed apoptotic cells express heat shock proteins on their plasma
membranes and dendritic cells are capable of distinguishing them from
nonstressed apoptotic cells. Here we provide evidence that when
purified heat shock protein 70 or chaperone-rich cell lysate (CRCL)
from syngeneic normal tissue is used as an adjuvant with nonimmunogenic
apoptotic tumor cells in vaccination, potent antitumor immunity can be
generated. This antitumor immunity is mediated by T cells because
antitumor effects are not observed in either severe combined
immunodeficiency or T cell-depleted mice. We further demonstrate that
vaccination of mice with apoptotic tumor cells mixed with liver-derived
CRCL as adjuvant were capable of enhancing the production of
TH1 cytokines, inducing specific cytotoxic T lymphocytes
and eliciting long-lasting antitumor immunity. Stress proteins from
autologous normal tissue components therefore can serve as danger
signals to enhance the immunogenicity of apoptotic tumor cells and
stimulate tumor-specific immunity
(Blood. 2003;101:245-252) It is well accepted that antigen-presenting
cells (APCs) efficiently acquire antigens from apoptotic tumor cells
and present them to T cells.1-4 However, the immunologic
consequences of this remain controversial.5,6 Macrophages
and dendritic cells (DCs) phagocytose apoptotic tumor cells through a
receptor-mediated pathway1-3 that results in tumor antigen
access to the cytoplasm and cross- presentation on APC major
histocompatibility complex (MHC) class I molecules.1,4
Therefore, apoptotic tumor cells induced by chemotherapy or
radiotherapy can theoretically be a suitable antigen source for
stimulation of antitumor responses. We have previously reported that
apoptotic 12B1-D1 (BCR-ABL+) leukemia cells can
be either immunogenic or nonimmunogenic in vivo depending on whether or
not these cells are heat stressed prior to apoptosis
induction.7 Furthermore, we have found that DCs are
capable of distinguishing stressed apoptotic tumor cells from
nonstressed ones.8 We have also demonstrated that stressed apoptotic tumor cells express heat shock proteins (HSPs) on their surface, which appear to play a critical role in enhancing their immunogenicity.7,8
Tumor-derived HSPs, also called chaperone proteins, when used as
vaccines, can induce protective immunity against their tumors of
origin.9 In our studies we found that vaccination with
multiple HSPs/chaperone proteins enriched from tumor lysate by free
solution isoelectric focusing (FS-IEF) induced specific antitumor
immunity in various tumor models.10,11 Some important
chaperone proteins such as glycoprotein 96 (gp96), HSP90,
HSP70, and calreticulum enriched by this method tend to complex
together.12 We refer to the FS-IEF-derived preparation as
chaperone-rich cell lysate (CRCL). We have previously reported that
tumor-derived CRCL has superior abilities to stimulate DCs compared to
purified individual HSPs such as HSP70 and gp96 (Y.Z. et al, manuscript
submitted), which have been used as tumor vaccines in mice and
humans.13,14
Several mechanisms have been proposed to explain how these HSP
complexes, which carry tumor-derived peptides as part of their chaperoning functions, can elicit immune responses.15-17
One possible hypothesis is that chaperone complexes supply both
antigens and danger signals to the immune system.15-17
These adjuvant effects/danger signals activate APCs, such as DCs,
leading to more efficient processing and presentation of HSP-chaperoned
peptides.18,19 We have previously reported that heat
stress induces HSP expression on the surface of apoptotic 12B1-D1 cells
and increases their immunogenicity.7,8 We therefore
reasoned that the immunogenicity of nonstressed apoptotic cells (which
do not express HSPs on their surface) may also be enhanced if an
exogenous source of HSPs is present at the vaccination site. To test
our hypothesis, normal syngeneic liver-derived chaperone proteins
(devoid of tumor-specific antigenic peptides) were coinjected with
nonstressed apoptotic tumors. This resulted in the reproducible
generation of durable and specific T cell-mediated antitumor immunity.
Nonstressed apoptotic cells alone or when combined with liver lysate,
generated by freeze-thaw and not enriched for chaperone complexes, were
ineffective vaccines. Therefore, we have demonstrated that normal
tissue (liver) CRCL may function as effective danger signals for the
immune system. FS-IEF is a simple and rapid method for the generation
of natural vaccine adjuvants active in enhancing immunogenicity of
apoptotic tumor cells resulting in the generation of potent antitumor immunity.
Mice
FS-IEF for chaperone enrichment and conventional purification
of HSP70
Induction of apoptotic cell death in 12B1-D1 cells To induce 12B1-D1 cells to undergo apoptosis, cells were treated with 40 nM AP2087 for 6 hours as described previously7 or with 100 µg/mL mitomycin C (Mit-C) for 1 hour. To confirm that 12B1-D1 cells were dying by apoptosis after Mit-C treatment, we performed annexin V-fluorescein isothiocyanate (FITC) and propidium iodide (PI) staining using the annexin-V-FLUOS staining kit (Roche, Indianapolis, IN) followed by flow cytometric analysis of cells. DNA fragmentation analysis was performed using the apoptotic DNA ladder kit from Roche.In vivo tumor growth experiments In the prophylactic model, mice were immunized with 2 × 106 Mit-C-treated cells, with or without 20 µg/mouse per vaccination of CRCL or HSP70, by subcutaneous injection into the left groin on days 14 and 7. On day 0, mice were
challenged with 2 × 104 (100% lethal dose
[LD100]) viable 12B1-D1 cells. Tumor size was measured
every other day with calipers once the tumors became palpable. Tumor
volume was calculated using the formula: length × width2 ×  /6. Differences in mean tumor volume
between groups were compared using an unpaired t test. Mice
with tumors were euthanized at the end points listed. Tumor-free mice
were kept for rechallenge experiments. In rechallenge experiments,
2 × 104 12B1-D1, 103 12B1, or
106 A20 leukemia cells (all LD100 doses
as determined by dose titration experiments, data not shown), were
injected into the right groin 56 to 80 days after the first challenge.
In the preestablished tumor model, mice were injected with 2 × 104 (LD100) viable 12B1-D1 cells in right groin on day 0. On day +1 or +3, mice were vaccinated as indicated by subcutaneous injection into the left groin. Tumor volume was measured at indicated time points. In depletion experiments, on days ELISPOT assay Enzyme-linked immunospot (ELISPOT) assays were performed to assess the interferon (IFN-) production of splenocytes from vaccinated mice following in vitro stimulation with Mit-C-treated apoptotic 12B1-D1 cells. Splenocytes/apoptotic cells (10:1 ratio) were
cocultured for 24 hours on Millipore MultiScreen-HA 96 well plates
(MAHA S45). The plates had been previously coated overnight with
anti-IFN- capture antibody (10 µg/mL, clone R4-6A2, rat mAb
antimouse IFN-; BD Pharmingen, San Diego, CA). Cells were then
washed out with copious amounts of PBS plus 0.05% Tween 20 (PBST).
Biotinylated anti-IFN- antibody (2 µg/mL, clone XMG1.2, rat mAb
antimouse IFN-; BD Pharmingen) was added for 2 hours. Free antibody
was washed out, and the plates were incubated with horseradish
peroxidase (HRP)-linked avidin (ABC Elite reagent, 1 drop each of
reagent A and reagent B/10 mL PBS; Vector Laboratories, Burlingame, CA)
for 1 hour, following extensive washing with PBST, and then washing
with PBS. Spots were visualized by the addition of the HRP substrate
3-amino-9-ethylcarbazole (AEC; Sigma Chemical, St Louis, MO) prepared
in acetate buffer (pH 5.0) with 0.015% hydrogen peroxide. Spots were
counted using a dissecting microscope. Wells of interest were
photographed with a microscope-mounted Cool SNAP CCD camera (RS
Photometrics, Tucson AZ), and images captured with RS Image, Version
1.07 (Roper Scientific, Tucson, AZ).
T-cell proliferation assays and bioassay to determine IL-2 production BALB/c mice were immunized with Mit-C-treated 12B1-D1 cells that were mixed with liver lysate, liver-derived HSP70, or liver CRCL (20 µg/mouse) on days14 and 7. For controls, mice were immunized
with an equal number of Mit-C-treated 12B1-D1 cells or 20 µg/mouse
liver-derived CRCL or saline. On day 2, splenocytes from the
immunized mice were harvested and cocultured with Mit-C-treated apoptotic 12B1-D1 cells. The ratio of splenocytes to apoptotic cells
was 10:1. After 72 hours of culture, the supernatant from each group
was collected and serially diluted in a 96-well plate. Interleukin 2 (IL-2)-dependent cytotoxic T lymphoid line 2 (CTLL-2) cells
were added to each well. Human recombinant IL-2 was used to generate a
standard curve. All assays were performed in triplicate wells. After 24 hours culture, [3H]-thymidine (1 µCi/well; 0.037 MBq) was added. For T-cell proliferation assays, splenocytes
were cocultured with apoptotic 12B1-D1 cells for 4 days before the
addition of [3H]-thymidine. The cells were harvested 18 hours later using a 96-well Packard cell harvester and the
radioactivity measured on a Packard beta counter.
Cytotoxicity assay BALB/c mice were immunized as indicated above. Five days after the second immunization, splenocytes from the immunized mice were harvested. The in vivo-primed splenocytes were cultured for 5 days with Mit-C-treated apoptotic 12B1-D1 cells. The ratio of splenocytes to apoptotic cells was 10:1. Stimulated effector cells were tested for cytolytic activity against 12B1-D1, parental 12B1, or A20 cells by nonradioactive cytotoxicity assay (Promega, Madison, WI) following the instructions provided. The percentage of cytotoxicity was determined according to the formula provided in the kit instructions.
CRCL from normal tissue enhances the immunogenicity of apoptotic 12B1-D1 tumor cells We have previously reported that 12B1-D1 leukemia cells undergo apoptosis in response to in vitro treatment with AP20187.7 A 6-hour exposure to 40 nM AP20187 induces apoptosis in more than 90% of 12B1-D1 cells, whereas 10% of cells remain clonogenic. When we inoculated mice with 5 × 105 AP20187-treated 12B1-D1 cells, rapid tumor growth occurred. This indicated that the apoptotic cells failed to induce an active immune response against the surviving clonogenic cells. To test whether syngeneic naive mouse liver-derived HSP70 or CRCL would provide adjuvant effects that may enhance the immunogenicity of AP20187-induced apoptotic 12B1-D1 tumor cells, we subcutaneously injected mice with AP20187-treated 12B1-D1 cells that were mixed with either liver-derived HSP70 or CRCL (20 µg/mouse, based on previous dose titration experiments, data not shown). We found that coinjection of HSP70 significantly delayed tumor growth compared with mice that were injected with AP20187-treated cells alone (Figure 1A). Moreover, CRCL provided superior adjuvant effects compared to HSP70, resulting in significant delay of tumor growth with rejection of tumors in 75% of mice (Figure 1A). In additional experiments, we compared the adjuvant effects of lipopolysaccharide (LPS) with those of liver-derived CRCL and found that they were comparable (Figure 1B), with both significantly delaying tumor progression when compared to no CRCL adjuvant.
We next investigated whether liver-derived CRCL would have similar
adjuvant effects in enhancing the immunogenicity of apoptotic 12B1-D1
cells induced by other agents, such as chemotherapy drugs. We used
Mit-C (100 µg/mL for 1 hour) to induce apoptosis in 12B1-D1 cells.
This resulted in more than 80% cells undergoing apoptosis within 6 hours as determined by annexin V-FITC/PI staining (Figure 2A). DNA from Mit-C-treated 12B1-D1
cells displayed electrophoretic ladder patterns typical of apoptotic
cells (Figure 2B). We then tested whether liver-derived CRCL can
enhance the immunogenicity of Mit-C-induced apoptotic 12B1-D1 cells.
To avoid release of cellular components from secondary necrosis that
occurs after apoptosis, we injected 12B1-D1 cells into mice immediately
following Mit-C treatment of the cells while 12B1-D1 cells maintained
their membrane integrity as determined by trypan blue exclusion and flow cytometry (data not shown). Compared to 6 hours of AP20187 treatment, 1 hour of treatment with 100 µg/mL Mit-C generated no
clonogenic cells. Mice were immunized on days
We have previously demonstrated that vaccination with CRCL derived from 12B1 (the parental cell line of 12B1-D1) elicited specific antitumor immunity (Y.Z. et al, manuscript submitted). 12B1-derived CRCL (presumably containing tumor antigenic peptides) has similar adjuvant effects to liver CRCL. Vaccination with either 12B1 tumor CRCL or liver CRCL as adjuvant plus apoptotic 12B1-D1 tumor cells induced potent antitumor immunity that protected 100% of mice from a lethal dose of 12B1-D1 challenge (Figure 3B). We further investigated whether vaccination with apoptotic 12B1-D1
cells plus CRCL as adjuvant could elicit therapeutic effects on
pre-established tumors. Mice received a lethal dose of 12B1-D1 on day
0, and were then immunized with Mit-C-treated apoptotic 12B1-D1 cells
plus CRCL as adjuvant in the opposite groin on day +1 or day +3.
Vaccination on day +1 with CRCL adjuvant-apoptotic tumor cells
significantly delayed tumor growth as compared with mock vaccination
(Figure 4). As expected, when vaccination
was delayed until day +3, tumor progression was suppressed but to a
lesser degree (Figure 4).
Immunity generated by CRCL adjuvant-apoptotic tumor cells is T-cell dependent We next investigated whether the antitumor response induced by apoptotic tumor cells with CRCL adjuvant was T cell dependent. SCID mice were subcutaneously injected with 5 × 105 AP20187 treated 12B1-D1 cells, with or without liver-derived CRCL adjuvant. Mice developed 12B1-D1 tumors at comparable rates in both groups (Figure 5A), suggesting that CRCL has no adjuvant effects in T cell-deficient mice. Moreover, tumor growth in mice coinjected with apoptotic cells plus LPS as adjuvant was not significantly delayed (Figure 5A).
To further evaluate the role of T-cell subsets in the immunity induced
by CRCL adjuvant plus apoptotic tumor cells, we performed in vivo T
cell-depletion experiments. CD4+, CD8+, or
both subsets of T cells were depleted by intraperitoneal injection with
200 µg anti-CD4 or anti-CD8 mAbs or both into mice on days Vaccination with CRCL adjuvant-apoptotic tumor cells induces tumor-specific CTLs Cell-mediated immunity, which is particularly important in suppressing tumors, is characterized by production of type I cytokines, activation of macrophages, and induction of cytotoxic T lymphocytes (CTLs). To explore whether vaccination with apoptotic tumor cells plus CRCL as adjuvant can induce type I cytokine secretion by T cells and generate tumor-specific CTLs, we examined IFN- and IL-2 production
as well as the cytolytic activities of splenocytes derived from
vaccinated mice. We found that vaccination with apoptotic tumor cells
plus CRCL adjuvant substantially increased the secretion of IL-2
(Figure 6A) and IFN- (Figure 6B) by
splenocytes on restimulation in vitro as measured by IL-2 bioassay and
ELISPOT assay, respectively. Moreover, spleen T-cell proliferation in
CRCL adjuvant-apoptotic tumor cell-vaccinated mice also increased
dramatically on restimulation with apoptotic tumor cells (Figure 6C),
compared to mock immunized mice. The induction of cytokine secretion or
T-cell proliferation was not due to nonspecific stimulation because
splenocytes from mice immunized with Mit-C-induced apoptotic cells
alone or liver CRCL alone produced limited amounts of cytokines and
those immunizations resulted in almost no T-cell proliferation (Figure
6). We also found that vaccination with apoptotic cells plus
liver-derived HSP70 as adjuvant induced IFN- and IL-2 secretion by
splenocytes, and increased T-cell proliferation, but at lower levels
when compared to CRCL as adjuvant (Figure 6). This may explain the
weaker in vivo protective effects following immunization with HSP70
adjuvant plus apoptotic 12B1-D1 cells induced by AP20187 (Figure 1A) or Mit-C (data not shown). Because both HSP70 and CRCL were derived from
whole liver lysate, we explored whether the liver lysate can also
confer adjuvant effects. We found that apoptotic cells coinjected with
liver lysate induced minimal cytokine production and T-cell
proliferation (Figure 6A-C).
CTLs are important in controlling tumor growth.22 Several
reports have shown that APCs can acquire antigens from apoptotic bodies
and can cross-prime CTLs in vitro.4,23 However, evidence that those apoptotic cells prime CTLs in vivo remains limited. We
therefore investigated whether or not vaccination with apoptotic tumor
cells plus CRCL as adjuvant induces measurable CTL activity. Splenocytes were collected from immunized mice and restimulated in
vitro with Mit-C-treated 12B1-D1 cells for 5 days and then tested for
cytolytic function against different tumor targets. Vaccination with
apoptotic cells plus CRCL elicited CTL activity (Figure
7A). In contrast, vaccination with
Mit-C-induced apoptotic tumor cells alone or with liver lysate failed
to generate CTL activity, whereas liver-derived HSP70 had less
impressive adjuvant effects in eliciting CTL activity (Figure 7A).
The 12B1-D1 cell line is derived from 12B1 by transfection of a plasmid encoding a death construct as described previously.7 12B1 cells therefore may contain all other tumor antigens of 12B1-D1 except the plasmid products. One of the advantages of using apoptotic cells as a vaccine is that they should contain the whole repertoire of tumor antigens. Theoretically, the CTLs induced by CRCL adjuvant plus apoptotic 12B1-D1 cells should also lyse 12B1 cells. We demonstrated that the CTL activity induced by CRCL adjuvant plus apoptotic 12B1-D1 cells was specific and potent enough to lyse the parental 12B1 targets (Figure 7B). Furthermore, we used another leukemia cell line, A20, as a target for CTLs to confirm the specificity of the CTL activity. Figure 7C shows that no lysis above background was detected when A20 cells were used as targets, confirming that the CTL activity induced by CRCL adjuvant-apoptotic cells was tumor specific. In summary, our data provide direct evidence that apoptotic leukemia cells, when combined with syngeneic tissue-derived CRCL as adjuvant, induced potent and specific CTLs in vivo. CRCL adjuvant-apoptotic tumor cells induce long-term, specific immunity Finally, we investigated whether the antitumor immunity induced by vaccination with CRCL adjuvant plus apoptotic tumor cells was long lasting. Mice surviving vaccination with either liver-derived or 12B1 tumor-derived CRCL adjuvant plus apoptotic 12B1-D1 cells were rechallenged with a lethal dose of 12B1-D1, 12B1, or A20 tumor cells 56 or 80 days after the initial challenge. All these mice rejected the rechallenge of 12B1-D1 tumor cells (Figure 8A). In contrast, age-matched naive mice developed 12B1-D1 tumor rapidly (Figure 8A). The long-term immunity also held true when mice were rechallenged with the parental 12B1 tumor (Figure 8B), but not when challenged with A20 B-cell leukemia (Figure 8C), confirming that the antitumor immunity induced by CRCL adjuvant plus 12B1-D1 apoptotic cells was long lasting, potent, and tumor-specific.
The deficiency of self/non-self paradigms led to new hypotheses proposed by Janeway24,25 and Matzinger26,27 to explain the immune response. The immune system is thought to react to "danger" associated with certain molecules of infectious organisms, or cell products released during tissue damage or stress.17 In addition to antigens that can be acquired, processed, and presented by APC, these danger signals are needed to activate local APCs, potentiating the immune responses against the antigens. In our studies, we found that 12B1-D1 leukemia cells, induced to undergo apoptosis by either AP20187 or Mit-C, were poorly immunogenic. In vivo immunization using these apoptotic cells induced no detectable antitumor immunity. In vitro, apoptotic 12B1-D1 cells had poor immunostimulatory activities on DCs in terms of inducing IL-12 production and enhancing imunostimulatory functions of DCs.8 Our results are consistent with other studies.28,29 Therefore, our data contribute the notion that apoptotic cells are, by default, nonimmunogenic to the immune system, because normal cell turnover must not induce active immune responses. Although apoptotic tumor cells can be efficiently taken up and their antigens presented by APCs,4,30 an active immune response is seldom generated in vivo because of lack of danger signals. However, when apoptotic cells are under stress, such as heat-stress apoptosis7 or pathogen-induced apoptosis,23,31,32 they are able to induce potent immune responses. How can the immune system recognize "stressful" apoptotic cells? We found that heat-stressed apoptotic 12B1-D1 cells expressed HSPs on their surface7 and these stressed apoptotic cells activated DCs.8 Because HSPs are emerging to be key danger signals to the immune system,17 we hypothesized that, by providing exogenous HSPs, the default (immune silent) pathway of apoptotic cells could be bypassed. To test our hypothesis, we purified HSP70 (constitutive form) and enriched CRCL from a syngeneic naive mouse liver. In vivo immunization with liver-derived CRCL provided no protection against subsequent autologous 12B1-D1 tumor challenge (data not shown). In addition, repeated injection of these syngeneic tissue components into BALB/c mice resulted in no apparent autoimmune phenomena (data not shown). However, when we coinjected mice with HSP70 and apoptotic tumor cells, antitumor immunity and specific CTLs were generated. We therefore demonstrated that HSP70 derived from naive mouse liver had adjuvant effects enhancing the immunogenicity of apoptotic tumor cells. Danger signals provided by HSP70, together with antigens from apoptotic tumor cells, induced specific antitumor immunity. Tumor-derived HSP70 has been used as a cancer vaccine against a variety of tumors.13,14 The antigen-chaperoning properties of HSP were highlighted in the tumor vaccine setting by the work of Srivastava.9 How does this soluble tumor-derived HSP70, with its chaperoned peptides, elicit strong cell-mediated antitumor immunity in the absence of adjuvants? Blachere et al showed for different peptide antigens (viral and nonviral CTL epitopes) that vaccination with gp96 or HSP70 induced a peptide-specific CTL response, whereas vaccination with peptides alone did not.33 Ciupitu et al reported that vaccination with HSP70/lymphocytic choriomeningitis virus (LCMV) peptide complexes elicited LCMV-specific CTLs and protective immunity against LCMV.34 Our results demonstrate that autologous naive mouse liver-derived HSP70, devoid of tumor antigens, can serve as an adjuvant to enhance the immunogenicity of apoptotic tumor cells. We have demonstrated that vaccination with tumor-derived CRCL induced
specific antitumor immunity against autologous tumor challenge in a
variety of mouse models.11,12 In all models, we found that
CRCL was generally more effective than purified individual HSPs in
generating antitumor immunity. We also demonstrated that tumor-derived
CRCL had a stronger ability to stimulate DCs than purified HSP70 or
gp96 (Y.Z. et al, manuscript submitted). Here, we have
documented that naive mouse liver-derived CRCL has superior adjuvant
effects when compared to HSP70 derived from the same tissue. This may
partially explain why tumor-derived CRCL is generally superior to
individual HSPs in terms of inducing antitumor
immunity.11,12 A secondary "danger signal" is
apparently important to activate APCs, which consequently activate
specific T cells. Why liver-derived CRCL provides better adjuvant
effects than does purified liver HSP70 is an area of active research in our laboratory. One possible explanation is that CRCL contains multiple
HSPs or chaperone proteins that synergistically activate APCs. Several
studies have documented that APCs bind and internalize gp96 through
receptor-mediated endocytosis, which leads to MHC class I-restricted
representation of gp96-chaperoned peptides and CTL
activation.35,36 The CD91 molecule
( Cell-mediated immunity, which is particularly important in suppressing
tumors, is characterized by production of type I cytokines, activation
of macrophages, and induction of CTLs. Previous reports have shown that
apoptotic cells are associated with induction of type II immune
suppressive cytokines, such as transforming growth factor CTLs are particularly important in tumor immunity.22 Several reports have shown that professional APCs can acquire antigens from apoptotic bodies and cross-prime CTLs in vitro.4,23 However, evidence that those apoptotic cells prime CTLs in vivo remains limited. We found that vaccination with CRCL adjuvant plus apoptotic tumor cells induced potent and specific CTLs, which appear to play an important role against 12B1-D1 tumor. The CTLs induced by vaccination with CRCL adjuvant plus apoptotic 12B1-D1 cells also lysed parental 12B1 cells. In fact, when we depleted CD8+ cells by using specific antibodies, we found that the antitumor immunity was partially abolished, suggesting that CD8+ T cells played an important role in tumor killing in vivo. This antitumor immunity was not abolished completely by depletion of CD8+ T cells alone, indicating that CD4+ T cells through a direct or indirect action contributed to the immune response. Accordingly, we found that depletion of CD4+ T cells significantly impaired the antitumor immunity, indicating that CTLs require CD4+ help. It is surprising that coinjection of liver lysate with apoptotic tumor cells did not induce antitumor immunity in vivo (data not shown). The pathologic necrotic cell death has been proposed to be dangerous to the immune system, because it releases cellular components, such as HSPs, mitochondria, double-strained DNA, among others, which may act as danger signals.29,45 It is possible that the lysate, generated simply by several cycles of freeze-thaw, cannot represent the true necrotic cell death occurring in pathologic conditions. In addition, the local concentrations of these "danger signals" released from dying cells may be important.17,46 It has been shown that tumor immunogenicity is associated with increased expression of HSP70 when tumor cells are undergoing necrotic death.47 Tumors that were genetically modified to express HSP or when exogenous HSP70 was provided during tumor cell killing decreased the immunosuppressive cytokine IL-10 expression.48 Furthermore, lysate from primary cells contains less HSP than their transformed counterparts and fail to mature DCs.46 CRCL, which contains at least a 20-fold of enrichment of major HSPs10,12 appears to provide a higher concentration of local danger signals. Currently, chemotherapy and radiotherapy remain the main treatment modalities for many cancers. Most of these therapies are thought to induce tumor cells to undergo apoptosis.49 These apoptotic tumor cells are attractive tumor antigen sources. However, without proper danger signals, they are largely ignored by the immune system, or may even induce tolerance.4,42,50 We have demonstrated that CRCL, enriched from normal or tumor tissues, provides potent adjuvant effects for enhancing the immunogenicity of apoptotic tumor cells that can induce potent, long-lasting antitumor immunity. Using FS-IEF, a relatively simple and rapid method, one can enrich large quantities of chaperone proteins from tissues in a less laborious and time-consuming manner compared to conventional purification of individual HSPs.10,12 These findings, together with the superior adjuvant effects, confer significant advantages of CRCL in terms of clinical applications.
The authors wish to thank Dr Douglas Lake for his helpful comments.
Submitted May 29, 2002; accepted August 7, 2002.
Prepublished online as Blood First Edition Paper, August 22, 2002; DOI 10.1182/blood-2002-05-1580.
Supported in part by the Leukemia and Lymphoma Society and the Tee up for Tots Fund.
The publication costs of this article were defrayed in part by page charge payment. Therefore, and solely to indicate this fact, this article is hereby marked "advertisement" in accordance with 18 U.S.C. section 1734.
Reprints: Emmanuel Katsanis, University of Arizona, Department of Pediatrics, 1501 N Campbell Ave, PO Box 245073, Tucson, AZ 85724; e-mail: katsanis{at}peds.arizona.edu.
1.
Albert ML, Pearce SF, Francisco LM, et al.
Immature dendritic cells phagocytose apoptotic cells via alphavbeta5 and CD36, and cross-present antigens to cytotoxic T lymphocytes.
J Exp Med.
1998;188:1359-1368
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Fadok VA, Warner ML, Bratton DL, Henson PM.
CD36 is required for phagocytosis of apoptotic cells by human macrophages that use either a phosphatidylserine receptor or the vitronectin receptor (alpha v beta 3).
J Immunol.
1998;161:6250-6257 3. Gregory CD. CD14-dependent clearance of apoptotic cells: relevance to the immune system. Curr Opin Immunol. 2000;12:27-34[CrossRef][Medline] [Order article via Infotrieve]. 4. Bellone M, Iezzi G, Rovere P, et al. Processing of engulfed apoptotic bodies yields T cell epitopes. J Immunol. 1997;159:5391-5399[Abstract]. 5. Restifo NP. Building better vaccines: how apoptotic cell death can induce inflammation and activate innate and adaptive immunity. Curr Opin Immunol. 2000;12:597-603[CrossRef][Medline] [Order article via Infotrieve]. 6. Lopes MF, Freire-de-Lima CG, DosReis GA. The macrophage haunted by cell ghosts: a pathogen grows. Immunol Today. 2000;21:489-494[CrossRef][Medline] [Order article via Infotrieve].
7.
Feng H, Zeng Y, Whitesell L, Katsanis E.
Stressed apoptotic tumor cells express heat shock proteins and elicit tumor-specific immunity.
Blood.
2001;97:3505-3512 8. Feng H, Zeng Y, Graner MW, Katsanis E. Stressed apoptotic tumor cells stimulate dendritic cells and induce specific cytotoxic T cells. Blood. In press. 9. Srivastava PK, Menoret A, Basu S, Binder RJ, McQuade KL. Heat shock proteins come of age: primitive functions acquire new roles in an adaptive world. Immunity. 1998;8:657-665[CrossRef][Medline] [Order article via Infotrieve].
10.
Graner M, Raymond A, Romney D, He L, Whitesell L, Katsanis E.
Immunoprotective activities of multiple chaperone proteins isolated from murine B-cell leukemia/lymphoma.
Clin Cancer Res.
2000;6:909-915 11. Graner MW, Zeng Y, Feng H, Katsanis E. Tumor-derived chaperone-rich cell lysates are effective therapeutic vaccines against a variety of cancers. Cancer Immunol Immunother. In press. 12. Graner M, Raymond A, Akporiaye E, Katsanis E. Tumor-derived multiple chaperone enrichment by free-solution isoelectric focusing yields potent antitumor vaccines. Cancer Immunol Immunother. 2000;49:476-484[CrossRef][Medline] [Order article via Infotrieve]. 13. Li Z. Priming of T cells by heat shock protein-peptide complexes as the basis of tumor vaccines. Semin Immunol. 1997;9:315-322[CrossRef][Medline] [Order article via Infotrieve]. 14. Przepiorka D, Srivastava PK. Heat shock protein-peptide complexes as immunotherapy for human cancer. Mol Med Today. 1998;4:478-484[CrossRef][Medline] [Order article via Infotrieve]. 15. Cho BK, Palliser D, Guillen E, et al. A proposed mechanism for the induction of cytotoxic T lymphocyte production by heat shock fusion proteins. Immunity. 2000;12:263-272[CrossRef][Medline] [Order article via Infotrieve]. 16. Heike M, Weinmann A, Bethke K, Galle PR. Stress protein/peptide complexes derived from autologous tumor tissue as tumor vaccines. B | |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
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Abstract
Introduction
2-macroglobulin receptor), which was initially
identified as a protein related to the low-density lipoprotein
receptor, has been recently shown to be a cell surface receptor for
gp96.
(TGF-