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tvi
ka*
*
Division of Experimental Immunology and Immunopathology, Department of Pathology, and
Department of Microbiology and Immunology, University of Louisville, Louisville, KY 40292;
Department of Pathology, Brigham and Women’s Hospital, Boston, MA 02115; and
§
Center for Blood Research and Department of Pathology, Harvard Medical School, Boston, MA 02115.
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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Mß2-integrin, that functions as an
adhesion molecule and a receptor for factor I-cleaved C3b, i.e., iC3b)
resulting in the priming of this iC3b receptor for cytotoxicity of
iC3b-opsonized target cells. This investigation explored mechanisms of
tumor therapy with soluble ß-glucan in mice. Normal mouse sera were
shown to contain low levels of Abs reactive with syngeneic or
allogeneic tumor lines that activated complement, depositing C3 onto
tumors. Implanted tumors became coated with IgM, IgG, and C3, and the
absent C3 deposition on tumors in SCID mice was reconstituted with IgM
or IgG isolated from normal sera. Therapy of mice with glucan- or
mannan-rich soluble polysaccharides exhibiting high affinity for CR3
caused a 57–90% reduction in tumor weight. In young mice with lower
levels of tumor-reactive Abs, the effectiveness of ß-glucan was
enhanced by administration of a tumor-specific mAb, and in SCID mice,
an absent response to ß-glucan was reconstituted with normal IgM or
IgG. The requirement for C3 on tumors and CR3 on leukocytes was
highlighted by therapy failures in C3- or CR3-deficient mice. Thus, the
tumoricidal function of CR3-binding polysaccharides such as ß-glucan
in vivo is defined by natural and elicited Abs that direct iC3b
deposition onto neoplastic cells, making them targets for circulating
leukocytes bearing polysaccharide-primed CR3. Therapy fails when tumors
lack iC3b, but can be restored by tumor-specific Abs that deposit iC3b
onto the tumors. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Early reports showed that ß-glucans functioned by stimulating host
defense mechanisms and were not toxic for tumors. ß-Glucans
stimulated macrophages (4), neutrophils (14),
and NK cells (15) to kill sensitive tumor cells. Some data
also suggested that ß-glucans could promote T cell-specific responses
(16), perhaps through triggering the secretion of IFN-
,
IL-6, IL-8, and IL-12 from macrophages, neutrophils, and NK cells
(17). A role for T cells in ß-glucan function was also
proposed because of absent tumoricidal activity in nude or T
cell-depleted mice (16, 18).
A role for CR3 in mediating responses to ß-glucan was first suggested
by research into the mechanisms of neutrophil phagocytosis of
iC3b-opsonized yeast. Stimulation of CR3-dependent phagocytosis or
degranulation was found to require the simultaneous ligation of two
distinct sites within CR3, one specific for iC3b and a second site
specific for yeast cell wall ß-glucans (19). Because
they lack cell-surface CR3-binding polysaccharides, erythrocytes
opsonized with iC3b were bound to neutrophils via CR3 but did not
stimulate phagocytosis or degranulation. More recently, a lectin domain
of CD11b (-subunit of CR3) was mapped to a C-terminal region located
distal to the I-domain binding sites for iC3b and ICAM-1 (20, 21). Soluble zymosan-derived polysaccharides (SZP) rich in
mannans or ß-glucans (SZPm or
SZPg) were shown to bind to CR3 with high
affinity, inducing a primed receptor state capable of triggering
phagocyte or NK cell cytotoxicity of iC3b-opsonized tumor cells that
otherwise did not trigger CR3-dependent responses
(22).
The sugar specificity of CR3 was found to be broader than was first
appreciated and included some polysaccharides containing mannose and
N-acetyl-D-glucosamine as well as
glucose (20). Although CR3 does not bind -mannans, data
reported with a ß-oligomannan that mediated attachment of
Candida albicans to phagocytes (23) via CR3
(24), as well as the finding of a mannose-rich SZP with
high affinity for CR3 (20), suggest that a less common
ß-mannan in some yeast also binds to CR3. Murine CR3 was recently
shown to have a sugar specificity and function similar to those of
human CR3 in priming phagocytes and NK cells for cytotoxicity of
iC3b-opsonized tumors (25).
This investigation focused on defining the in vivo mechanisms of tumoricidal activity induced by therapy with soluble CR3-binding polysaccharides such as ß-glucan. Using mouse tumor models, C3 deposition onto tumors was shown to be primarily regulated by tumor-reactive Abs that directed complement activation. This targeting of tumor cells with opsonic iC3b was an essential factor in determining the specific cells to be eliminated in ß-glucan therapy. The molecular requirements for ß-glucan-mediated tumoricidal activity were defined in tests with C3- and CR3-deficient mice. It was concluded that the specificity of the cell-mediated cytotoxicity mediated by ß-glucans is regulated by tumor-specific Abs that target only tumor cells with iC3b, and this spares normal tissues that are not opsonized with iC3b.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The BALB/c (H-2d) mammary tumor line Ptas64 was provided by Dr. Wei-Zen Wei (Michigan Cancer Foundation, Wayne State University, Detroit, MI). The MMT (H-2k) mammary tumor line and the sarcoma 180 (S-180) (MHC class I-negative) line were obtained from the American Type Culture Collection (ATCC, Manassas, VA). The C57BL/6-derived (H-2b) EL-4 lymphoma line was from Dr. Nia-Kong Cheung (Sloan-Kettering Cancer Institute, New York, NY). These cell lines were maintained in RPMI 1640 containing 2 mM L-glutamine, antibiotics, and 10% FCS. The MC38 adenocarcinoma and MB49 bladder carcinoma lines (both H-2b) were provided by Dr. Donald W. Kufe (Harvard University, Boston, MA) and maintained in DMEM containing 10% FCS, antibiotics, and 4.5 g/L glucose. The rat hybridomas secreting TIB211 IgM anti-mouse CD8 mAb and M1/70 IgG anti-mouse CD11b (CR3) were from the ATCC. Mouse 11C1 hybridoma secreting IgG2a mAb specific for murine mammary tumor virus (MMTV) (26) was generously provided by Dr. Hiroshi Fuji (Department of Molecular Immunology, Roswell Park Cancer Institute, Buffalo, NY). Each mAb was isolated from ascites fluid generated in BALB/c mice, and portions of each were coupled to biotin or FITC using standard methods. Biotinylated rat anti-mouse Ly-6G (Gr-1, anti-granulocyte) mAb, anti-mouse CD3-FITC, PE-labeled rat anti-mouse CD80 (B7-1), anti-mouse NK cell (DX5), both unlabeled IgG and IgG-PE, and streptavidin-PE were from PharMingen (San Diego, CA). Goat F(ab')2 anti-mouse IgG-FITC and anti-mouse IgM-FITC were from Southern Biotechnology Associates (Birmingham, AL), and goat F(ab')2 anti-mouse C3-FITC was from Cappel (Durham, NC).
SZP
Methods for isolation and characterization of SZP, a soluble low
molecular mass (
10 kDa) polysaccharide consisting primarily of
mannose or glucose, were previously reported (20).
Briefly, polysaccharides are eluted from zymosan by heating to 95°C
in 90% formic acid for 20 min. After discarding large polysaccharides
precipitating with 70% ethanol, a small polysaccharide fraction is
isolated by precipitation with 80% ethanol. After digestion with
-mannosidase and removal of anionic proteins by Mono-Q fast protein
liquid chromatography, a uniform-sized soluble polysaccharide is
isolated by chromatography with S-200HR. As reported recently
(25), there has been some variation in the size and
mannose/glucose composition of such SZP preparations that is dependent
on the starting batch of yeast. SZP preparations used in this
investigation either consisted almost entirely of mannose and are
referred to as SZPm (20), or
consisted primarily of ß-1,3-glucans and are referred to as
SZPg (25). The CR3-binding affinity
of 125I-labeled SZPm
preparations have been 5-fold higher than preparations of
125I-SZPg, and thus,
whereas optimal priming of mouse or human CR3 for cytotoxic activation
in response to iC3b-opsonized tumor cells in vitro required 1 µg
hexose per ml of SZPm (22),
comparable CR3 priming with SZPg required 5
µg/ml of hexose (25). Contaminating LPS was removed from
SZPm to levels below the limit measurable with
the Limulus lysate test (E-Toxate, Sigma, St. Louis, MO)
using Detoxi Gel (Pierce, Rockford, IL). With
SZPg, all detectable LPS was removed using Triton
X-114 (27).
Mice
Female, 4–16-wk-old, normal and SCID BALB/cJ mice, as well as normal C57BL/6 mice, were purchased from The Jackson Laboratory (Bar Harbor, ME). Mice deficient in C3 (C3-/- on a 129/J background) (28), CR1/2 (Cr2-/-; 129/J) (29), or CR3 (CD11b-/-; 129/J) were previously described (30). The C3- and CR3-deficient mice and their wild-type littermates were bred and maintained at Harvard Medical School until shipped to the University of Louisville for tumor therapy experiments. For some experiments as noted, the CR3-deficient trait was transferred onto a BALB/cH background (subscript "H" denoting Harvard Medical School origin of BALB/c mice) by eight or nine backcrosses to normal BALB/cH mice, so that age- and sex-matched BALB/cH wild-type and CR3-deficient littermates were available for tumor therapy experiments.
Purification of normal IgM and IgG reactive with the Ptas64 tumor cell line
Gammaglobulins from 50 ml of normal BALB/c mouse serum (Harlan Bioproducts for Science, Indianapolis, IN) were precipitated with 50% saturated ammonium sulfate, dialyzed into 10 mM Tris/HCl pH 7.5, and chromatographed on a 1.6 x 10-cm Mono-Q fast protein liquid chromatography column (Amersham Pharmacia Biotech, Piscataway, NJ) at 10 ml/min. Bound proteins were eluted with a linear salt gradient over 60 min, in which the limit buffer was 0.5 M NaCl in 10 mM Tris/HCl, pH 7.5. Fractions enriched for IgM and/or IgG Abs reactive with Ptas64 cells were identified by indirect immunofluorescence staining with anti-IgM-FITC or anti-IgG-FITC and flow cytometry. IgM and IgG Abs were isolated by S-300HR (Amersham Pharmacia Biotech) chromatography using a 1.6 x 50-cm column. The IgM fraction, 2.0 ml and 2.62 mg/ml of IgM (determined by ELISA; see below), stained 30% of Ptas64 cells by indirect immunofluorescence at a dilution of 1:20. Based on similar staining activity of the starting serum diluted 1:4, this represented a 40% yield of the IgM Abs. The IgG fraction of 4 ml had comparable staining activity of Ptas64 cells when diluted 1:50, representing a 20% yield of IgG Abs. Each Ig fraction was depleted of detectable LPS using Triton X-114 and sterilized by ultrafiltration with a 0.22 µm filter.
ELISA for IgM
Immulon-2 plates (Dynatech, Chantilly, VA) were coated with affinity-purified anti-mouse IgM (Boehringer Mannheim Biochemicals, Indianapolis, IN) for capture of mouse IgM test samples or MOPC-104E IgM (Sigma) tested in parallel to establish a standard curve. Bound IgM was detected with anti-mouse IgM-peroxidase (Boehringer Mannheim Biochemicals), followed by Sigma 104 phosphate substrate solution (Sigma). The OD490 of each well was measured with a Rainbow MicroELISA plate reader (SLT Instruments, Salzburg, Austria).
Mouse tumor models for SZP therapy
Mice were implanted s.c. in a mammary fat pad with 1–5 x 106 Ptas64, MMT, or MC38 cells freshly harvested from culture and suspended in 100–200 µl of PBS. Small, palpable tumors were allowed to form in all mice over 10–14 days before initiation of daily i.p. or i.v. (tail vein) injections of 200 µg SZPm or SZPg (100 µl, 2 mg hexose/ml of PBS), with a control group of mice receiving daily i.v. injections of 100 µl PBS. After 9–14 days of therapy (same time for all mice in an individual experiment), the mice were sacrificed, and tumors were removed, trimmed of surrounding tissue, and weighed.
Flow cytometry analysis of tumors
Single-cell suspensions of the freshly removed tumors were generated by gently teasing small tissue fragments between forceps in HBSS (Life Technologies, Grand Island, NY), followed by filtration through a cell strainer (Falcon Division, Becton Dickinson Labware, Franklin Lakes, NJ). Large debris was removed by layering cell suspensions over 3 ml of heat-activated FCS and allowing sedimentation at 1 x g for 10 min in an ice bath. After two washes with ice-cold RPMI 1640, the cells were suspended in RPMI 1640 and maintained on ice. Immunofluorescence staining of 5 x 105 cells was conducted on ice using standard techniques, including the addition of propidium iodide to exclude dead cells from analysis (31). An EPICS Profile II (Coulter Electronics, Miami Lakes, FL) was used for flow cytometry, and the listmode data were analyzed with WinList 3.0 (Verity Software House, Topsham, ME).
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Tests of normal sera from BALB/c, C57BL/6, and 129/J mice
demonstrated naturally occurring IgM and IgG Abs reactive with
syngeneic and allogeneic tumor cell lines (Table I
). The presence of these Abs explained
the ability of these tumor lines to activate complement with resultant
C3 deposition, because no C3 was detectable on tumors cells incubated
with fresh serum from SCID mice (not shown). The only exception among
these tumors was the EL-4 lymphoma, in which levels of IgM and IgG
tumor-reactive Abs in C57BL/6 sera (Table I) were too low to mediate
detectable C3 deposition (not shown). Previous in vitro studies had
shown that the density of C3 deposited onto Ptas64 cells by normal
BALB/cJ serum was adequate to trigger the
cytotoxic activation of the ß-glucan-primed CR3 expressed by
neutrophils, macrophages, or NK cells (25).
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. Young
BALB/c (Table I
) or 129/J mice (not shown) had lower levels of such
tumor-reactive Abs than did older mice, and tumors removed from young
mice exhibited less staining for Ig and C3 than did tumors removed from
older mice (Fig. 1, A and B). Amounts of IgM and
IgG tumor-reactive Abs increased with age in
BALB/cJ (Table I) and 129/J (not shown) mice,
reaching plateau levels at 
3 mo of age. As expected, no
tumor-reactive Abs were detected in SCID BALB/cJ
mice (Table I), and tumors removed from SCID mice exhibited no staining
for IgM, IgG, or C3 (Fig. 1C). Reconstitution of SCID mice
with i.v. normal IgM or IgG generated in vivo opsonization of implanted
tumors with C3 (Fig. 1D). Sera from 129/J mice deficient in
either serum C3 (C3-/-) or B cell CR1/2
(Cr2-/-) exhibited levels of tumor-reactive IgM
and IgG Abs that were similar to those of normal 129/J mice (Table I).
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BALB/cJ mice with Ptas64 mammary tumors were
tested for a therapeutic response to daily injections of
SZPm given either i.p. or i.v. for 2 wk (Fig. 2). This experiment with 18 mice was
repeated twice with similar results and then was repeated three more
times with LPS-free SZPm. Because of variations
in mean tumor weight in the PBS control groups (0.7–1.3 g), tumor
weight reduction in each experiment was determined by comparison only
with the PBS control group from the same experiment. Overall, in six
experiments, mice given i.p. SZPm exhibited less
tumor reduction (45% ± 10%; p < 0.001) than did
mice receiving i.v. SZPm (90% ± 7.2%;
p < 0.001). Similar results obtained with LPS-free
SZPm indicated that minor LPS contamination did
not explain the tumoricidal response to SZPm. In
each experiment, one or two mice of six in the i.v.
SZPm group had no detectable tumor.
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SZPg therapy of young (5-wk-old)
BALB/cJ mice implanted with Ptas64 tumors
resulted in less tumor reduction (
50%; not shown) than with older
(2-mo-old) BALB/cJ mice (90%; Fig. 2). To
determine whether this was due to a reduced amount of tumor-reactive
Abs in the younger mice that resulted in less C3 deposition on tumor
cells, the amount of IgG and C3 on tumors in the young
BALB/cJ mice was augmented by passive
immunization with an IgG2a mAb to MMTV, a tumor Ag expressed on Ptas64
cells. Initial experiments to determine the optimal dose of the
anti-MMTV mAb indicated that 200 µg of the IgG2a mAb given on
alternate days for 2 wk resulted in maximum deposition of C3 onto tumor
cells. When this mAb therapy was combined with daily i.v.
SZPg in young BALB/cJ mice
implanted with Ptas64 tumors, the increased amount of C3 on tumors was
associated with a significantly greater tumor reduction in response to
SZPg (Fig. 3).
Administration of the anti-MMTV mAb resulted in increased
deposition of IgG and C3 on breast tumors (Fig. 3, top).
However, despite this greatly increased complement activation by
tumors, treatment with i.v. anti-MMTV alone had no significant
effect on tumor growth. A therapeutic benefit with the i.v.
anti-MMTV mAb was only observed when it was combined with i.v.
SZPg (Fig. 3, bottom).
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Previous reports on the tumoricidal activity of ß-glucans in
mice had suggested a requirement for T cells because of therapy
failures in nude (18, 32), thymectomized (6),
or normal mice depleted of T cells with anti-CD4 and anti-CD8
(16). As expected, T cell-deficient nude BALB/c (not
shown) and SCID BALB/c (Table I) mice lacked detectable Abs reactive
with Ptas64 tumor cells, and Ptas64 tumors removed from nude (not
shown) or SCID BALB/c (Fig. 1C) mice did not exhibit
staining for Ig or C3. Reconstitution of SCID mice i.v. with normal IgM
or IgG resulted both in C3 opsonization of tumors (Fig. 1D)
and in a significant tumor reduction response to
SZPg therapy (Fig. 4). Although tumor reduction with
combined therapy was less than had been observed with normal BALB/c
mice (Fig. 2), the proportions of tumor cells bearing C3 were also
comparably less. With reconstitution of IgM tumor-reactive Abs to serum
levels comparable with normal BALB/c mice, only 10% of tumor cells
exhibited C3 staining, whereas with IgG reconstitution to normal serum
IgG Ab levels, 25% of tumor cells were stained for C3 (Fig. 4).
However, with IgG-reconstituted SCID mice, a tumor reduction of 50%
was considerably less than the 90% tumor reduction observed in normal
BALB/c mice (Fig. 2), in which 50–75% of tumor cells exhibited
staining for C3 (Fig. 1).
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Previous in vitro experiments had shown that SZP-mediated
cytotoxicity required SZP priming of both leukocyte CR3 and iC3b on the
tumor cells (25, 33). A correlation of tumor C3 density
with resultant SZP-mediated tumor reduction was also suggested by
current experiments with mice given Abs that enhanced the density of C3
on tumors (Figs. 3 and 4). The requirement for C3 in SZP therapy was
confirmed in experiments with C3-deficient 129/J mice implanted with
the MMT mammary carcinoma. SZPm therapy of normal
129/J mice resulted in tumor reduction (Fig. 5; p < 0.01) similar to
that of normal BALB/c mice (Fig. 2), and flow cytometry of tumors
showed abundant deposition of C3 on >80% of cells (not shown). By
contrast, in C3-deficient 129/J mice, there were no significant tumor
reduction (Fig. 5) and no C3 on tumors (not shown), but the relative
amount of IgG staining on tumors did not differ from that of the normal
129/J mice.
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). As expected,
significant tumor reduction in response to SZPg
therapy occurred only with normal (56.5 ± 10%, p
< 0.001) and not with CR3-deficient mice (Fig. 5). However, unlike in
BALB/cJ mice, sera from both the normal and
CR3-deficient BALB/cH mice exhibited lower levels
of IgG Abs reactive with Ptas64 cells (Table I), and as a result, fewer
cells from tumors were stained with anti-IgG-FITC or C3-FITC. There
was no difference in serum IgM tumor-reactive Abs or staining of
excised tumor cells with anti-IgM-FITC. However, with tumors from
both normal and CR3-deficient BALB/cH mice, only
26% of cells stained for IgG, and only 29% of cells stained for C3.
By contrast, with Ptas64 tumors removed from
BALB/cJ mice, a mean of 78% of cells stained for
IgG and 61% stained for C3 (Fig. 1). Thus, the reduced
therapeutic response in normal BALB/cH vs
BALB/cJ mice appears to have been due to lower
levels of naturally occurring IgG tumor-reactive Abs that translated
into a reduced surface density of C3 on tumor cells. No significant
differences in IgG- or C3-specific staining were detectable on tumors
removed from normal vs CR3-deficient mice. Humoral response to Ptas64 tumor cells in tumor-bearing mice
The sensitivity of Ptas64 tumors to SZP therapy appeared to
correspond to serum levels of naturally occurring Abs to Ptas64 that
regulated levels of C3 deposition onto tumors. The increase in
tumor-reactive IgG Abs in 1–3-mo-old BALB/cJ
mice, as well as the variation between BALB/c mice at the same age but
raised in different environments (i.e., BALB/cJ
vs BALB/cH), further suggested that these Abs
represented an elicited response rather than natural Abs that are
primarily IgM. To determine whether Ptas64 cells might stimulate an
immune response in BALB/c mice, sera from BALB/cJ
mice implanted with Ptas64 tumors for 6 wk were examined for levels of
IgM and IgG tumor-reactive Abs (Fig. 6).
Sera from mice given i.v. SZPg were compared with
sera from mice in an i.v. PBS control group. The results indicated that
6 wk of tumor implantation stimulated a 3- to 4-fold increase in
tumor-reactive IgG Abs, whereas there was no increase in IgM Ab levels.
However, mice receiving SZPg did not exhibit a
greater increase in Ab than mice in the PBS control group (Fig. 6).
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Previous in vitro tests had shown that neutrophils, macrophages,
and NK cells bearing SZP-primed CR3 were each capable of killing
iC3b-opsonized Ptas64 cells (25). Flow cytometry of
tumors from mice treated with SZP vs PBS found similar proportions of
NK cells (15%), granulocytes (25%), macrophages (25%),
CD8+CD3+ T cells (10%),
and CD8-CD3+ T cells
(15%). There was also no evidence for increased proportions of the
minor subset of
CR3+CD8+ T cells (2% of
T cells) in tumors or spleens from these mice vs non-tumor-bearing
mice. Moreover, the proportions of these major leukocyte subsets were
the same in tumors removed from normal vs CR3-deficient mice. This
shows that the reduced tumoricidal activity in CR3-deficient mice was
not due to reduced leukocyte infiltration of tumors, suggesting that
leukocyte diapedesis into such implanted s.c. tumors is normal in
CR3-deficient mice.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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It is well known that ß-glucan responses occur only in certain strains of mice bearing specific tumors. Our data suggest that reports of the sensitivity or resistance of specific tumors to ß-glucan corresponds to the presence or absence of Abs capable of opsonizing the tumor with iC3b. Over the last 39 years, S-180 has been the most frequently used tumor to screen for ß-glucan tumoricidal function (3, 34, 35, 36). Not only were Abs to S-180 found in sera from several mouse strains, but also S-180 was found to lack MHC class I that can suppress CR3-mediated NK cell cytotoxicity (37). Moreover, we confirmed (38) that C57BL/6 sera contain very low levels of Abs reactive with the syngeneic (H-2b) EL-4 lymphoma, a finding consistent with the resistance of EL-4 tumors to ß-glucan therapy (39). However, when a mAb to GD2 ganglioside was used in combination with C57BL/6 serum to opsonize the GD2+ EL-4 cells, the cells exhibited abundant staining for C3 and were killed efficiently by ß-glucan-primed neutrophils (J. Yan and G. D. Ross, unpublished observation). By contrast, with another H-2b tumor, the MC38 adenocarcinoma, the presence of Abs in normal 129/J (H-2b) sera produced in situ opsonization of tumors with iC3b and allowed ß-glucan therapy to be successful.
Naturally occurring Abs present in both BALB/c and 129/J sera produced in situ deposition of C3 on tumors, making them sensitive to SZP therapy. The presence of naturally occurring IgG Abs suggests that these are not "natural Abs" per se that are usually IgM, but are probably elicited Abs to environmental Ags that cross-react with tumor Ags. Similar levels of tumor-reactive Abs were found in sera from C3- and CR1/2-deficient mice, even though CR1/2-deficient mice have a limited repertoire of natural Abs, and both types of complement deficiency lead to a reduced response to T cell-dependent Ags (29, 40). Moreover, mammary tumors implanted into BALB/c mice elicited an increased level of tumor reactive IgG. These findings have a human counterpart, because normal human sera contain IgM and IgG Abs reactive with human mammary tumor lines, and likewise, levels of tumor-reactive IgG are elevated in patients with breast cancer (33). Similar findings of natural and elicited Abs to murine (38, 41) or human (42, 43) mammary carcinoma have been reported. Our studies suggest that although such humoral responses by themselves are inadequate to mediate tumor rejection, the resulting C3 deposition targets the tumor cells for cytotoxic recognition by leukocytes bearing ß-glucan-primed CR3.
This investigation showed an important role for tumor-reactive Abs in facilitating a response to ß-glucan. Passively administered normal IgG was more effective than normal IgM in opsonizing tumors in SCID mice, and the best C3 opsonization of tumors was achieved in mice given a mAb to the MMTV tumor Ag. However, it is noteworthy that the greatly increased complement activation mediated by this mAb had virtually no effect on tumor growth. This emphasizes the inherent resistance of tumors to complement-mediated cytotoxicity and shows why passively administered mAbs to tumor Ags or vaccines that generate a good humoral response are frequently ineffective therapeutically. Nevertheless, the enhanced response to ß-glucan in mice given Abs to tumor Ags, particularly SCID mice, shows the potential efficacy of passive mAb therapy in combination with ß-glucan in cancer patients who may be immunosuppressed.
Although the tumors used in this study required surface C3 for
recognition by CR3-bearing leukocytes, other tumors may express natural
ligands for ß-glucan-primed CR3. For example, SZP-primed human NK
cells mediate CR3-dependent cytotoxicity of unopsonized K562 cells
(22), and SZP-primed murine NK cells mediate CR3-dependent
cytotoxicity of unopsonized YAC-1 cells (V. Vtvika,
unpublished observation). Moreover, there have been several reports
showing a tumoricidal activity of ß-glucan-treated macrophages with
unopsonized tumor cell line targets in vitro (44, 45, 46).
Previous investigators had proposed that CTL were essential for ß-glucan tumoricidal activity because therapy was unsuccessful in either nude (18, 32) or T cell-depleted mice (16). However, the tumor reduction induced in SCID mice reconstituted only with IgG argues against a necessary role for CTL in ß-glucan therapy. Moreover, the absence of MHC class I on S-180 sarcoma that has been used frequently as a tumor model for ß-glucan therapy argues further against an essential role for CTL. Nevertheless, with other tumors that express tumor-specific peptides on class I, a role for ß-glucan in augmenting Ag presentation cannot be excluded. In particular, the enhanced CR3-dependent functions of macrophages, and possibly also dendritic cells, may enhance tumor Ag uptake and presentation to T cells. Furthermore, polysaccharide-dependent activation of macrophage CR3 stimulates the secretion of several cytokines, particularly IL-12 (17, 47). By contrast, in the absence of ß-glucan therapy, IL-12 secretion might even be suppressed within iC3b-opsonized tumors that mediated CR3-dependent monocyte adhesion without stimulating toxicity. For example, monocyte IL-12 secretion is suppressed by ligation of CR3 to iC3b-opsonized erythrocytes that lack CR3-priming polysaccharides (48).
Similar proportions of CR3+ granulocytes, macrophages, and NK cells were noted in tumors from mice treated with SZP vs PBS, suggesting that SZP therapy does not lead to a specific recruitment of leukocytes into tumors. The inflammatory response resulting from Ab-dependent activation of complement with release of C3a and C5a should occur equally in tumors from SZP-treated mice vs PBS control mice, and this may serve to recruit CR3+ granulocytes and macrophages. Each of these leukocytes, as well as NK cells, are capable of CR3-dependent tumoricidal activity in vitro (25).
The unpredictable outcome of adoptive therapy with BRMs has limited their use in cancer. Although ß-glucans have been viewed as adjuvants that could enhance immune responses, the current data indicate that ß-glucans function through naturally occurring or elicited Abs by providing a cell-mediated effector mechanism that is normally functional only against microbial pathogens. Furthermore, in BALB/c mice with Ptas64 tumors, ß-glucan did not enhance levels of tumor-reactive Abs above those observed in mice receiving PBS. With this recognition that tumors that lack opsonizing C3 will not respond to ß-glucan therapy, it would be preferable to restrict the use of ß-glucans to patients known to have tumor-reactive Abs through active or passive immunization.
Although SZP therapy of BALB/c mice bearing Ptas64 tumors resulted in
an average tumor weight reduction of 90%, some therapy failures also
occurred. These were particularly noted in several mice in one
experiment in which i.v. SZP therapy was continued for up to 6 wk (data
not shown). Examination of the tumors from these mice showed that
80% of the tumor cells were not stained by anti-IgG- or
anti-C3-FITC, despite an increase in serum levels of tumor-reactive
IgG. These data suggest that SZP therapy can function to select mutant
cells that are tumor Ag negative and thus resistant to SZP therapy
because they are no longer opsonized with Ab and C3. Tumor escape
through immune selection for tumor Ag-negative cells is a well-known
confounding factor in immunotherapy.
ß-Glucans can potentially be used to generate a novel cell-mediated effector mechanism for tumor vaccines and mAbs to tumor Ags that otherwise rely mostly on the direct cytotoxic action of C. This therapy appears to have the greatest applicability to metastatic tumors that have lost MHC class I and thus have escaped recognition by CTL (49, 50). Such metastatic tumors frequently express polysaccharide or ganglioside tumor Ags for which there is an array of available vaccines and mAbs. Although the complement activation stimulated by such mAbs is inefficient when it relies solely on the complement membrane attack complex, it might be adequate to mediate the iC3b-opsonization of tumors required for cytotoxicity by leukocytes bearing ß-glucan-primed CR3. Finally, if ß-glucan therapy were combined with vaccines that stimulated both Abs and immune CTL, then the likelihood for immunologic escape would be further reduced, because it would require tumors to lose both MHC class I and all class II-dependent tumor Ags.
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Acknowledgments |
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ová and Ms. Jana
Vtviková. |
Footnotes |
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2 Current address: Department of Internal Medicine, Yale University School of Medicine, 333 Cedar Street, LCI 610, New Haven, CT 06520-8031.
3 Address correspondence and reprint requests to Dr. Gordon D. Ross, Department of Pathology, University of Louisville, Louisville, KY 40292. E-mail address:
4 Abbreviations used in this paper: BRM, biologic response modifier; iC3b, factor I-cleaved C3b fragment of C3; MMTV, murine mammary tumor virus; SZP, soluble zymosan-derived polysaccharide made up primarily of either mannan (SZPm) or ß-glucan (SZPg); S-180, sarcoma 180.
Received for publication May 11, 1999. Accepted for publication June 29, 1999.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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tvika, M. Pitman, R. C. Goldman, G. D. Ross. 1996. Analysis of the sugar specificity and molecular location of the ß-glucan-binding lectin site of complement receptor type 3 (CD11b/CD18). J. Immunol. 156:1235.[Abstract]
tvika, V., B. P. Thornton, G. D. Ross. 1996. Soluble ß-glucan polysaccharide binding to the lectin site of neutrophil or NK cell complement receptor type 3 (CD11b/CD18) generates a primed state of the receptor capable of mediating cytotoxicity of iC3b-opsonized target cells. J. Clin. Invest. 98:50.[Medline]
tvika, J. Yan, M. Haniková, T. N. Mayadas, G. D. Ross. 1999. The ß-glucan-binding lectin site of mouse CR3 (CD11b/CD18) and its function in generating a primed state of the receptor that mediates cytotoxic activation in response to iC3b-opsonized target cells. J. Immunol. 162:2281.tvika, V., B. P. Thornton, T. J. Wieman, G. D. Ross. 1997. Targeting of NK cells to mammary carcinoma via naturally occurring tumor cell-bound iC3b and ß-glucan-primed CR3 (CD11b/CD18). J. Immunol. 159:599.[Abstract]
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