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*
Department of Biochemistry, University of Illinois at Chicago, Chicago, IL 60680; and
The Ben May Institute for Cancer Research, the Committee on Immunology, and the Department of Pathology, University of Chicago, Chicago, IL 60637
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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as well as to exert
an anti-MOPC-315 CTL effect. The preferential importance of the
B7-2 molecule may be due to the higher level of B7-2 than of B7-1
expression on B220+ cells and on tumor cells from the s.c.
tumor nodule of low dose L-PAM-treated MOPC-315 tumor
bearers and the selective up-regulation of the B7-2 molecule in the
draining of these mice. Thus, the B7-2 molecule plays a dominant role
in the acquisition of T cell-dependent tumor-eradicating immunity in
low dose L-PAM-treated mice bearing a large MOPC-315 tumor,
suggesting that one of the mechanisms by which chemotherapy may enhance
antitumor immunity is through up-regulation of critical costimulatory
molecules that enhance antitumor responses. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The ligands for CD28 receptor belong to the B7 family, which consists of at least two members, the B7-1 (CD80) (3) and the B7-2 (CD86) (4, 5). The B7-1 and B7-2 molecules are expressed by multiple cell types including B cells, macrophages, dendritic cells, and T cells (6, 7, 8, 9). However, the expression of the B7-1 and B7-2 costimulatory molecules is regulated independently, and differences exist in the temporal expression of the two molecules following stimulation (2, 6, 7). In addition, differences have been noted in the kinetics of binding of the B7-1 and B7-2 molecules to the CD28 (10). Moreover, several studies have shown the preferential importance of B7-1 or B7-2 molecule for the development of a particular immune response (11, 12, 13, 14, 15, 16, 17). For example, Lenschow et al. (16) have demonstrated that engagement of the B7-2 (and not the B7-1) molecule is critical for the development of diabetes in nonobese diabetic mice, while Miller et al. (15) have demonstrated the importance of the B7-1 (and not the B7-2) molecule during the clinical relapsing phase of murine experimental allergic encephalomyelitis. Based on these studies from nontumor systems (11, 12, 13, 14, 15, 16, 17), it has been suggested that the B7-2 molecule is the dominant costimulatory ligand in initiating primary immune responses, while the B7-1 molecule (which is up-regulated later during the immune response (6, 7)), is more critical in prolonging primary immune responses or costimulating secondary immune responses (2, 5).
In spite of the intense research into the importance of the endogenously expressed B7-1 and/or B7-2 molecule for the in vivo development of an immune response in a variety of nontumor systems (11, 12, 13, 14, 15, 16), only a few studies have been performed to elucidate the importance of the "naturally" expressed B7 molecules for the development of antitumor immunity (i.e., in response to tumor cells not transfected with the B7 genes). Moreover, the information gathered is limited to the importance of the B7-1 and/or B7-2 molecule for the development of antitumor immunity in normal mice (18, 19, 20), with no information about the importance of the B7-1 and/or B7-2 molecule in the acquisition of T cell-dependent tumor-eradicating immunity by hitherto immunosuppressed animals bearing large tumors.
The current studies were undertaken to determine whether the
"naturally" expressed B7-1 and/or B7-2 molecule is important for
the acquisition of T cell-dependent tumor-eradicating immunity by mice
bearing a large tumor that were subjected to a therapeutic modality
known to lead to the acquisition of T cell-dependent
tumor-eradicating immunity. More specifically, we assessed the
effect of anti-B7-1 or anti-B7-2 mAb treatment on the
curative effectiveness of low dose L-phenylalanine
mustard (L-PAM)5
for MOPC-315 tumor bearers under conditions in which the chemotherapy
depends on the acquisition of potent CD8+ T cell-mediated
tumor-eradicating immunity by the hitherto immunosuppressed tumor
bearers (21, 22, 23, 24). Our studies revealed the preferential importance of
the B7-2 molecule for the curative effectiveness of low dose
L-PAM for MOPC-315 tumor bearers. Moreover, our studies
have shown that the B7-2 molecule plays a dominant role in the low dose
L-PAM-induced acquisition of the ability of TILs from
MOPC-315 tumor bearers to produce IFN- and IL-2, as well as exert a
potent anti-MOPC-315 CTL activity. Finally, our studies suggest
that the preferential importance of the B7-2 molecule for the curative
effectiveness of low dose L-PAM for MOPC-315 tumor bearers
is due to the fact that the chemotherapy leads to the selective
expression of the B7-2 molecule in the draining lymph nodes of MOPC-315
tumor bearers and/or the higher level of B7-2 (relative to B7-1)
expression on B220+ cells and tumor cells from the tumor
nodules of low dose L-PAM-treated MOPC-315 tumor
bearers.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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We have used the MOPC-315 plasmacytoma, which was maintained in
vivo in female BALB/c mice 7 to 10 wk old (Charles Rivers Breeding
Laboratories, Wilmington, MA). Routinely, mice were inoculated s.c.
with 1 x 106 viable tumor cells, a dose that is
at least 300-fold higher than the minimal lethal tumor dose and that
leads to the appearance of a palpable tumor in 4 to 5 days. Thereafter,
the tumors grow progressively, killing the mice in 
16 days.
L-PAM therapy
A fresh stock solution of L-PAM (Sigma Chemical
Co., St. Louis, MO) was prepared as previously described (24). A dose
of 1.75 to 2.5 mg of L-PAM per kg of body weight (low dose)
was administered i.p. to BALB/c mice bearing a large (20-mm) tumor
that resulted from the s.c. inoculation of 1 x 106
MOPC-315 tumor cells 10 days earlier. The doses of drug used are
curative for most BALB/c mice bearing a large MOPC-315 tumor, and they
require the participation of CD8+ T cell-dependent
antitumor immunity in tumor eradication (22, 24). Significant
regression of the s.c. tumor nodules is evident as early as 4 or 5 days
after the chemotherapy (21, 22, 23, 24).
Anti-B7-1 or anti-B7-2 mAb treatment
Affinity-purified hamster IgG anti-B7-1 (hybridoma 16-10A1) (25) or rat IgG2a anti-B7-2 (hybridoma GL-1) (26) mAb was used throughout the studies to block the in vivo expression of the B7-1 or B7-2 molecule, respectively. The treatment protocol consisted of an i.p. injection of 100 to 200 µg of anti-B7-1 or anti-B7-2 mAb given 2 h after the administration of low dose L-PAM to MOPC-315 tumor bearers and was followed by the injection of 100 to 200 µg of the same mAb 2 and 4 days later. As control, low dose L-PAM-treated MOPC-315 tumor bearers were given normal rat IgG (NIgG; Sigma Chemical Co.).
Flow cytometric analysis
Single-cell suspensions of viable cells derived from the tumor nodules and draining lymph nodes of untreated mice or low dose L-PAM-treated MOPC-315 tumor-bearing mice were prepared as previously described (22, 23). The expression of the B7-1 and B7-2 costimulatory molecules on these cells was assessed by indirect immunofluorescence staining utilizing biotinylated anti-B7-1 (16-10A1) or anti-B7-2 (GL-1) mAb followed by phycoerythrin (PE)-conjugated streptavidin (all of which were purchased from PharMingen, San Diego, CA). Since the relative fluorescence intensity of cells exposed to biotinylated NIgG (PharMingen) plus PE-conjugated streptavidin varied among the samples obtained from the same site on different days after the chemotherapy, we adjusted for the relative fluorescence intensity of cells exposed to NIgG plus PE-conjugated streptavidin for all such groups. In experiments analyzing the expression of B7-1 and B7-2 molecules on large vs small cells among cells derived from the tumor nodules, we gated on the cells of interest on the basis of light scatter properties. In experiments assessing the phenotypes of the cells expressing up-regulation of B7-1 and B7-2 molecules, cells were stained simultaneously for B7 expression (with biotinylated anti-B7-1 or anti-B7-2 mAbs followed by PE-conjugated streptavidin) and for the expression of B220, MAC-1, or CD3 surface molecules (by the use of the appropriate FITC-conjugated mAb, all of which were purchased from PharMingen). Finally, flow cytometric analysis of 20,000 viable cells was conducted on a Coulter EPICS Elite ESP (Coulter Electronics, Hialeah, FL). The results obtained in the single-color determinations with regard to B7 expression are presented as histograms. The results obtained in the two-color determinations are presented as fold increase in the mean fluorescence intensity (MFI) for B7-1 or B7-2 staining on the B220+, MAC-1+, or CD3+ cells. The fold increase in the MFI represents the fold increase in mean fluorescence intensity of cells exposed to biotinylated anti-B7-1 or anti-B7-2 mAb plus PE-conjugated streptavidin over that of cells exposed to biotinylated NIgG plus PE-conjugated streptavidin (27).
TIL preparations
TILs were obtained as previously described (22, 23) from the
s.c. tumor nodules of mice treated with low dose L-PAM
plus NIgG, or plus anti-B7-1 or anti-B7-2 mAb. In studies
assessing the effect of anti-B7-1 or anti-B7-2 mAb treatment on
low dose L-PAM-induced up-regulation of cytokine production
at the tumor site, the TILs were obtained on day 3 after the
chemotherapy, since this is the earliest time after the chemotherapy at
which the TILs consistently produce detectable levels of IFN- and
IL-2. In studies assessing the effect of anti-B7-1 or anti-B7-2
mAb treatment on low dose L-PAM-induced acquisition of
TIL-mediated anti-MOPC-315 CTL activity at the tumor site, the TILs
were obtained on day 4 after the chemotherapy, since this is the
earliest time after the chemotherapy at which the TILs consistently
exhibit significant levels of anti-MOPC-315 CTL activity (22, 23).
Finally, at both time points, TIL preparations from mice treated with
L-PAM plus anti-B7-1 or anti-B7-2 mAb did not
contain a percentage of CD4+ or CD8+ cells
significantly different than TIL preparations from mice treated with
L-PAM plus NIgG.
Positive selection of CD8+ TILs
CD8+ TILs derived from the s.c. tumor nodules of mice treated 4 days earlier with low dose L-PAM were purified with the aid of biotinylated anti-CD8 mAb, streptavidin-conjugated magnetic beads, and MiniMacs Separation Columns (Miltenyi Biotec Inc., Sunnyvale, CA), according to the manufacturer’s recommendation. The purity of the positively selected CD8+ TIL preparations from mice treated with low dose L-PAM plus anti-B7-2 or plus NIgG was >90%, as determined by flow cytometric analysis.
Cytokine production
TILs were seeded at a concentration of 6 x
106 cells/well in 24-well plates (Falcon, Becton
Dickinson Labware, Lincoln Park, NJ) in 2 ml of DMEM (Life Technologies
Inc. Gaithersburg, MD) supplemented with 5% FBS, 5 x
10-5 M 2-ME (Sigma Chemical Co.), 50 U/ml penicillin, 50
µg/ml streptomycin, and 15 mM HEPES buffer (Life Technologies Inc.).
The TILs were then incubated for 24 h in the absence of additional
tumor cells, because their in vivo stimulation with tumor Ag and/or
residual Ag present in the TIL preparations from mice treated 3 days
earlier with low dose L-PAM was sufficient to trigger the
production of significant levels of IL-2 and IFN-. To rule out the
possibility that reduced cytokine production is due to insufficient
stimulation by tumor cells, we added PMA (Sigma Chemical Co.) at a
final concentration of 10 ng/ml to the plated TILs in some experiments.
Such an approach was undertaken since PMA without ionomycin was shown
to enhance the lysis of target cells by alloimmune splenic T cells (28)
or by freshly isolated splenic T cells from low dose
L-PAM-treated MOPC-315 tumor bearers (29) in an Ag-specific
manner.
Cytokine quantification
The level of IL-2 and IFN- secreted by the TILs into the
culture medium was determined by an ELISA. For the IL-2 ELISA, we used
rat anti-mouse IL-2 mAb JES6-1A12 as capture Ab and biotinylated
rat anti-mouse IL-2 mAb JES6-5H4 as detection Ab (PharMingen). For
the IFN- ELISA, we used rat anti-mouse IFN- mAb R46A2
(American Type Culture Collection, Rockville, MD) as capture Ab and
biotinylated rat anti-mouse IFN- mAb XMG1.2 (PharMingen) as
detection Ab. The sensitivity of the ELISA for IL-2 is 15 pg/ml, and
that for IFN- is 20 pg/ml.
Antitumor cytotoxicity assay
Freshly isolated TILs were evaluated for their anti-MOPC-315 lytic activity, as previously described (22, 23), by a 3.5-h 51Cr release assay in which three to four different E:T ratios were used. The percentage of specific 51Cr release was calculated by the formula [(Ecpm - Scpm)/(Mcpm - Scpm)] x 100, where Ecpm represents the 51Cr released by target cells incubated with effector cells, Scpm represents the spontaneous release, and Mcpm represents the maximal release obtained by the addition of 2% Nonidet P-40 detergent (Particle Data Corp., Elmhurst, IL) solution. The results are presented as mean ± SE 51Cr released by TILs from 9 to 12 individual mice per treatment group. In addition, the mean of the levels of cytotoxicity at all E:T ratios of a particular treatment group were converted to LU per 1 x 107 effector cells, wherein 1 LU is defined as the number of effector cells producing 20% lysis of 1 x 104 target cells.
Statistical analysis
To determine the significance of differences in the fraction of mice surviving following different treatments, the generalized Savage (Mantel-Cox) test was used. For all other statistical analyses, Student’s t test was used. A p value of 0.05 or lower was considered significant in both tests.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Low dose L-PAM therapy of mice bearing a large
MOPC-315 tumor was previously shown to lead to the acquisition of
potent T cell-dependent tumor-eradicating immunity that in turn leads
to the cure of most mice (21, 22, 23, 24). In this study, experiments were
performed to determine the importance of the B7-1 or the B7-2 molecule
for the curative effectiveness of low dose L-PAM as an
indicator for the importance of these molecules for the acquisition of
tumor-eradicating immunity by the tumor bearers. Specifically, we
assessed the effect of blocking the B7-1 or the B7-2 costimulatory
molecule (through the use of anti-B7-1 or anti-B7-2 mAb) on the
survival time of mice bearing an 20-mm s.c. MOPC-315 tumor and
treated with low dose L-PAM. As seen in Figure 1
, administration of anti-B7-2 mAb
drastically reduced the curative effectiveness of 1.75 mg/kg
L-PAM for MOPC-315 tumor bearers with only 10% of the mice
remaining alive and tumor free at the end of the 60-day observation
period as compared with 70% of the mice surviving following the
administration of low dose L-PAM in conjunction with NIgG.
In contrast, administration of anti-B7-1 mAb did not reduce
significantly the percentage of MOPC-315 tumor bearers cured with low
dose L-PAM. However, the tumor regressed more slowly in
mice that were cured following treatment with low dose
L-PAM plus anti-B7-1 mAb than in mice that were cured
following treatment with low dose L-PAM plus NIgG (Fig. 2), with complete tumor regression
observed in 13.5 ± 0.8 vs 10.6 ± 0.3 days, respectively
(p 
0.05).Thus, the B7-2 molecule plays a
dominant role, while the B7-1 molecule plays only a secondary role, in
the curative effectiveness of low dose L-PAM for MOPC-315
tumor bearers.
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Since CD28/B7-2 engagement was found to be more important than
CD28/B7-1 engagement for the curative effectiveness of low dose
L-PAM for MOPC-315 tumor bearers, experiments were
performed to determine whether B7-2 is selectively up-regulated after
the chemotherapy in the tumor nodules of MOPC-315 tumor bearers.
Specifically, we studied the B7-1 and B7-2 expression in the tumor
nodules of untreated tumor-bearing mice or tumor-bearing mice treated 2
to 3 days earlier with low dose L-PAM. Since more than one
population of cells was evident in the tumor nodules of low dose
L-PAM-treated MOPC-315 tumor bearers based on light scatter
properties, we analyzed the data for the effect of low dose
L-PAM on B7-1 and B7-2 expression separately on large cells
and on small cells. As seen in Figure 3,
up-regulation of B7-1 and B7-2 expression by the large cells as well as
by the small cells was observed on days 2 and 3 after the chemotherapy.
Studies were conducted next to identify the cell type(s) that
up-regulated B7-1 and/or B7-2 expression in the tumor nodules of low
dose L-PAM-treated MOPC-315 tumor bearers. Specifically,
expression of B7-1 and B7-2 molecules was examined on
B220+, MAC-1+, and CD3+ cells among
the small cells and among the large cells from the tumor nodules of
untreated mice and mice treated 3 days earlier with low dose
L-PAM. As seen in Figure 4,
low dose L-PAM therapy was associated with up-regulation of
both B7-1 and B7-2 expression on B220+, MAC-1+,
and CD3+ cells. However, while B7-1 expression was higher
than B7-2 expression on MAC-1+ cells, B7-2 expression was
higher than B7-1 expression on B220+ cells and on
CD3+ cells. In addition, B7-1 and B7-2 expression was
up-regulated on large cells that did not express B220, MAC-1, or CD3.
These cells appeared to include MOPC-315 tumor cells based on light
scatter properties (data not shown). Interestingly, the MFI for B7-2
expression on "tumor cells" from low dose L-PAM-treated
MOPC-315 tumor bearers was substantially higher (threefold) than the
MFI for B7-1 expression. Thus, although low dose L-PAM
therapy is associated with up-regulation of both B7-1 and B7-2
molecules in the tumor nodules of MOPC-315 tumor bearers, all of the
cell types examined, with the exception of MAC-1+ cells,
express higher levels of B7-2 than of B7-1 molecules.
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In light of reports illustrating the importance of the
draining lymph nodes for the induction of antitumor immunity (19, 30),
experiments were conducted to elucidate the effect of low dose
L-PAM therapy on B7-1 and B7-2 expression in the
draining lymph nodes of MOPC-315 tumor bearers. As seen in Figure 5, low dose L-PAM therapy led
to the selective up-regulation of B7-2, but not B7-1, expression. The
up-regulation in B7-2 expression was evident on both day 2 and day 3
after chemotherapy. In an attempt to gain some insight into the effect
of L-PAM on the expression of B7-1 and B7-2 molecules in
lymph nodes that are not exposed to MOPC-315-associated Ags, we
conducted such determinations with lymph nodes from normal mice because
the MOPC-315 tumor is highly metastatic and lymph nodes that are
distant from the s.c. tumor nodule are also exposed to
MOPC-315-associated Ags (31). Our studies revealed no up-regulation of
the expression of either the B7-1 or B7-2 molecule in lymph nodes of
normal mice injected with low dose L-PAM 2 or 3 days
earlier (data not shown), indicating that exposure to tumor cells (or
tumor-associated Ags) is also required to induce up-regulation of B7-2
expression in the lymph nodes of mice injected with
L-PAM.
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, low dose L-PAM therapy was
associated with up-regulation of B7-2 expression on B220+,
MAC-1+, and CD3+ cells in the draining lymph
nodes of MOPC-315 tumor bearers, with B220+ cells
demonstrating the greatest increase in B7-2 expression.
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production by TILs from low dose
L-PAM-treated MOPC-315 tumor bearers
Since IL-2 is the primary cytokine produced as a consequence of
CD28 ligation (32) and this cytokine is important for T cell
proliferation, we determined the effect of anti-B7-1 or
anti-B7-2 mAb treatment on IL-2 production by TILs derived from the
s.c. tumor nodule of low dose L-PAM-treated MOPC-315
tumor bearers. More specifically, we determined the level of IL-2 in
culture supernatants of TILs derived from the s.c. tumor nodules of
MOPC-315 tumor bearers that were treated 3 days earlier with low dose
L-PAM in conjunction with anti-B7-1 mAb, anti-B7-2
mAb, or NIgG. As seen Figure 7
(top), anti-B7-2 mAb treatment, but not
anti-B7-1 mAb treatment, led to a significant reduction in IL-2
production by TILs derived from the tumor nodules of mice treated with
low dose L-PAM.
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production by TILs from MOPC-315 tumor bearers since 1) low
dose L-PAM therapy of MOPC-315 tumor bearers was previously
shown to lead to up-regulation of IFN- production at the tumor site
(33, 34) and 2) neutralization of IFN- production by mAb was
previously shown to reduce the curative effectiveness of low dose
L-PAM for MOPC-315 tumor bearers (34). As seen in Figure 7
(bottom), anti-B7-2 mAb treatment, but not
anti-B7-1 mAb treatment, led to a significant reduction in IFN-
production by TILs derived from the tumor nodules of low dose
L-PAM-treated MOPC-315 tumor bearers.
Finally, to assure that the reduced production of IFN- by TILs from
mice that were treated with L-PAM plus anti-B7-2 mAb is
not due to insufficient stimulation by tumor cells, we added PMA to
such cultures. Specifically, we compared the level of IFN-
production following exposure to PMA of TILs from mice treated with
L-PAM plus anti-B7-2 mAb relative to TILs from mice
treated with L-PAM plus NIgG or L-PAM plus
anti-B7-1 mAb. As a reference point, we assessed simultaneously the
level of IFN- produced by TILs from the three treatment groups when
cultured in the absence of PMA. As seen in Figure 8, TILs from mice treated with
L-PAM plus NIgG or L-PAM plus anti-B7-1 mAb
(but not L-PAM plus anti-B7-2 mAb) secreted a
significantly higher level of IFN- when cultured in the presence, as
compared with the absence, of PMA. In addition, when the TILs were
cultured in the presence of PMA, TILs from mice treated with
L-PAM plus anti-B7-2 mAb, but not TILs from mice
treated with L-PAM plus anti-B7-1 mAb, produced
significantly less IFN- than TILs from mice treated with
L-PAM plus NIgG. In fact, the magnitude of reduction in the
level of IFN- production as a consequence of anti-B7-2 mAb
administration to low dose L-PAM-treated MOPC-315 tumor
bearers was essentially the same when the TILs were cultured in the
presence or the absence of PMA (i.e., to 50% of the level produced
by TILs from mice treated with L-PAM plus NIgG). Thus, the
B7-2 molecule (and not the B7-1 molecule) plays an important role in
the acquisition of the ability of TILs from low dose
L-PAM-treated MOPC-315 tumor bearers to secrete IL-2 and
IFN-.
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Since low dose L-PAM therapy was previously shown
to lead to a massive accumulation of CD8+ T cells with
acquired anti-MOPC-315 CTL activity in the s.c. tumor nodules of
hitherto immunosuppressed MOPC-315 tumor bearers (22), experiments were
conducted to determine the relative importance of B7-1 and B7-2
molecules for this process. Specifically, we assessed the effect of
anti-B7-1 or anti-B7-2 mAb administration to low dose
L-PAM-treated MOPC-315 tumor bearers on the level of
anti-MOPC-315 CTL activity exhibited by their TILs. As seen in
Figure 9, administration of
anti-B7-2, but not anti-B7-1, mAb to low dose
L-PAM-treated MOPC-315 tumor bearers led to a substantial
reduction in the anti-MOPC-315 lytic activity exhibited by their
TILs. Moreover, when the data were converted to LU, TILs from mice
treated with L-PAM plus anti-B7-2 mAb exhibited 30%
of the lytic activity exhibited by TILs from mice treated with
L-PAM plus NIgG or L-PAM plus anti-B7-1 mAb
(i.e., 125 vs 400 or 417 LU, respectively). Finally, purified
CD8+ TILs from mice treated with L-PAM plus
anti-B7-2 mAb were also inferior to purified CD8+ TILs
from mice treated with L-PAM plus NIgG in terms of their
anti-MOPC-315 lytic activity (e.g., they led to 22.5 ± 1.9 vs
44.9 ± 2.2% 51Cr release at an E:T ratio of 1:1).
Thus, the B7-2 molecule (and not the B7-1 molecule) plays an important
role in the acquisition of CTL activity by TILs from low dose
L-PAM-treated MOPC-315 tumor bearers.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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production. Similarly,
Nakajima et al. (13) have shown that anti-B7-2 mAb inhibits the
production of autoantibody of the IgG1 and IgG2b (the production of
which is dependent on type 2 cytokines) but not the IgG2a (the
production of which is not dependent on type 2 cytokines) subclass
in a murine model for systemic lupus erythematosus and prevents the
development of nephritis. These studies led to the conclusion that the
B7-1 molecule induces the production of type-1 cytokines, while the
B7-2 molecule induces the production of type-2 cytokines (2, 11, 12, 13).
Consequently, it was proposed that the preferential importance of the
B7-1 or the B7-2 molecule in the development of a particular immune
response is a function of whether the particular immune response is Th1
or Th2 dependent (13).
However, not all observations made with regard to the preferential
dependence of a particular immune response on the endogenously
expressed B7-1 or B7-2 molecule fit the above model. For example,
anti-B7-2, and not anti-B7-1, mAb was shown to block allograft
rejection (16) as well as the development of diabetes in nonobese
diabetic mice (14), even though these responses depend on type 1
cytokines. Here we extend these latter observations by demonstrating
that the B7-2 molecule plays a dominant role (while the B7-1 molecule
plays only a secondary role) in the curative effectiveness of low dose
L-PAM for MOPC-315 tumor bearers, under conditions that
depend on type 1 cytokine production (34, 35). Moreover, we provide
information that the B7-2 molecule is actually involved in the low dose
L-PAM-induced acquisition of the ability of TILs from
MOPC-315 tumor bearers to produce IL-2 and IFN- and exert a CTL
response.
The observations that only anti-B7-2 mAb treatment, which reduced
the curative effectiveness of low dose L-PAM for MOPC-315
tumor bearer, also led to a substantial reduction in the ability of
TILs from low dose L-PAM-treated mice to produce IL-2 and
IFN- as well as to exert CTL activity are consistent with the
interpretation that anti-B7-2 mAb-mediated reduction in these
functions is responsible at least in part for the reduced curative
effectiveness of low dose L-PAM. Support for such an
interpretation is provided by our previous observations illustrating
the importance of IFN- (34) and CTL (22, 23, 36) for the curative
effectiveness of low dose L-PAM for MOPC-315 tumor bearers.
In addition, IL-2 production, although not evaluated directly, most
likely plays an important role in the acquisition of T cell-mediated
tumor-eradicating immunity given that IL-2 is important for T cell
proliferation. Thus, although the ability of TILs from
L-PAM-treated MOPC-315 tumor bearers to produce IL-2 and
IFN- as well as to exert CTL activity was reduced, but not
abrogated, as a consequence of anti-B7-2 mAb treatment, the
concurrent reduction in all three immune functions most likely played
an important role in reducing the overall tumor-eradicating potential
of the TILs.
The fact that low dose L-PAM therapy which depends on B7-2 expression for its curative effectiveness for MOPC-315 tumor bearers is associated with selective up-regulation of B7-2 expression in the draining lymph nodes of MOPC-315 tumor bearers suggests that up-regulation of B7-2 expression in the draining lymph nodes may be important for the induction of the generation of T cell-dependent tumor-eradicating immunity following low dose L-PAM therapy. Other studies support the concept that the draining lymph nodes play an important role in the generation of T cell-mediated antitumor immunity (19, 30). For example, Yang et al. (19) reported recently that endogenous expression of both the B7-1 and B7-2 molecules in the draining lymph nodes contributes to the priming of T cells from normal mice against P815 tumor cells inoculated s.c. Furthermore, treatment of the mice with anti-L-selectin (MEL-14) mAb completely prevented the appearance of P815-primed CD8+ T cells in the draining lymph nodes of these mice, resulting in enhanced growth of the s.c. P815 tumors. However, even if the induction of the immune response occurs in the draining lymph nodes, the maturation/amplification of the T cell-dependent tumor-eradicating immunity is still likely to occur in the s.c. tumor nodules of these mice and B7 expression at the tumor site can play an important role in this process (18, 19). Thus, the observed up-regulation of B7 expression in the tumor nodules of low dose L-PAM-treated MOPC-315 tumor bearers is consistent with such a scenario.
Although low dose L-PAM therapy leads to up-regulation of both B7-1 and B7-2 expression in the tumor nodules of low dose L-PAM-treated MOPC-315 tumor bearers, these molecules are up-regulated differently on different cell types. Specifically, B7-1 expression is higher than B7-2 expression on MAC-1+ cells, while B7-2 expression is higher than B7-1 expression on B220+, CD3+, and apparently also on MOPC-315 tumor cells. These observations are consistent with the observations of Baskar et al. (18) demonstrating that rejection of MHC class II-transfected SaI tumor cells by normal mice is associated with up-regulation of B7-1 and B7-2 expression on both host cells and tumor cells and B7 blockade prevents the tumor rejection. Although the study by Baskar et al. did not address the issue of the importance of up-regulation of B7 expression on host cells for the acquisition of antitumor immunity, their study implicated the up-regulation of B7 expression on tumor cells as important for the acquisition of antitumor immunity. Host cells that display up-regulation of B7 expression are also likely to be important for the acquisition of tumor-eradicating immunity in light of a recent report by Huang et al. (37) illustrating that most of the CTL priming in normal mice inoculated with B7-1-transfected colon carcinoma cells expressing influenza nucleoprotein is done by host APCs (although B7 expression on host APCs was not studied). Taken together, it is likely that both tumor cells and host APCs that display up-regulation of B7 expression contribute to the acquisition of tumor-eradicating immunity.
One apparent discrepancy between the observations of Baskar et al. (18) and our observations is that while both B7-1 and B7-2 molecules were equally effective in the acquisition of tumor-eradicating immunity following the inoculation of MHC class II-transfected SaI tumor cells, B7-2 expression was more important than B7-1 expression for the acquisition of tumor-eradicating immunity following low dose L-PAM therapy of MOPC-315 tumor bearers. The reason for this discrepancy is unknown at present. However, the fact that MOPC-315 cells from mice treated 2 to 3 days earlier with low dose L-PAM express much higher levels of B7-2 than B7-1 molecules, while MHC class II-transfected SaI tumor cells express comparable levels of B7-1 and B7-2 molecules on day 2 after tumor inoculation into normal mice, may help explain the difference in the relative importance of B7-1 and B7-2 molecules for the acquisition of tumor-eradicating immunity in the two tumor systems. In addition, the fact that B7-2 molecule is up-regulated more than B7-1 on B220+ cells in the tumor nodule of low dose L-PAM-treated MOPC-315 tumor bearers as well as in the draining lymph nodes of these mice may also contribute to the greater importance of the B7-2 than of the B7-1 molecule in the MOPC-315 tumor system.
At present, it is not known whether B7-2 expression on tumor cells and/or on host cells is important for the acquisition of CTL activity by TILs from low dose L-PAM-treated MOPC-315 tumor bearers. If we assume that B7 expression on tumor cells from low dose L-PAM-treated MOPC-315 tumor bearers is important for the acquisition of TIL-mediated CTL activity, our observations would appear to be in conflict with reports by Gajewski et al. (20) that the B7-1 and not the B7-2 molecule on tumor cells efficiently stimulates CTL generation. However, as acknowledged by Gajewski et al., the superiority of B7-1+ tumor cells over B7-2+ tumor cells in inducing CTL activity and protective immunity is limited to the use of irradiated tumor cells (20). Moreover, as pointed out by these investigators, previously activated CD8+ T cells may respond equally to stimulation by irradiated B7-1+ or irradiated B7-2+ tumor cells in contrast to the hypothetically more stringent requirement of naive cells for B7-1 expression. Thus, since in our system 1) live tumor cells from low dose L-PAM-treated MOPC-315 tumor bearers express elevated levels of the B7-2 molecule and 2) MOPC-315 tumor growth is associated with priming of the T cells before the administration of low dose L-PAM (38), B7-2-expressing tumor cells should be capable of costimulating CTL generation. In other words, it is not surprising that the B7-2 molecule is effective in costimulating CTL generation in low dose L-PAM-treated MOPC-315 tumor bearers. What is surprising is that B7-1 is not as effective as B7-2 in doing so; this may be a manifestation of the lower level of B7-1 expression on the tumor cells and/or on B220+ cells in the tumor nodule of low dose L-PAM-treated MOPC-315 tumor bearers and/or the lack of B7-1 expression in the draining lymph nodes of these mice.
In addition to the up-regulation of B7-2 expression as a consequence of low dose L-PAM therapy of MOPC-315 tumor bearers, the chemotherapy is also associated with a shift in the cytokine profile at the tumor site from cytokines with inhibitory activity for CTL generation (33, 39) to cytokines that can promote CTL generation (34). One of the cytokines that is up-regulated at the tumor site following the chemotherapy is TNF (34), which is critical for the curative outcome of low dose L-PAM for MOPC-315 tumor bearers (35). Interestingly, among the many functions of TNF, TNF was recently found to lead to selective up-regulation of B7-2 (and not B7-1) expression on stimulation cultures of tumor-infiltrated spleen cells from MOPC-315 tumor bearers (40). In light of these observations and since low dose L-PAM-induced up-regulation of TNF production precedes the up-regulation of B7-2 expression at the tumor site, it is conceivable that TNF may also play a role in the selective up-regulation of B7-2 expression in low dose L-PAM-treated MOPC-315 tumor bearers.
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2 Address correspondence and reprint requests to Dr. Margalit B. Mokyr, Department of Biochemistry (M/C 536), The University of Illinois at Chicago, 1819 West Polk Street, Chicago, IL 60612.
3 In partial fulfillment of the requirements for the Doctor of Philosophy Degree.
4 Supported in part by the Dorothea Fleming Cancer Research Fellowship Award.
5 Abbreviations used in this paper: L-PAM, L-phenylalanine mustard; TILs, tumor-infiltrating lymphocytes; NIgG, normal IgG; MFI, mean fluorescence intensity; LU, lytic unit which corresponds to the number of effector cells producing 20% lysis of 1 x 104 target cells; PE, phycoerythrin.
Received for publication March 13, 1997. Accepted for publication October 29, 1997.
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in the therapeutic outcome. Cancer Immunol. Immunother. 41:363.[Medline]
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) mAb on the
survival time of mice bearing a large (20-mm) s.c. MOPC-315 tumor and
treated with low dose L-PAM (1.75 mg/kg). As a
reference point, we provide information regarding the survival time of
tumor-bearing mice treated with 1.75 mg/kg L-PAM plus NIgG
(
). Numbers in parentheses indicate the number of mice alive and
tumor free at the end of the observation period relative to the number
of mice subjected to the treatment protocol. * indicates a
statistically significant (p 
) and mice
treated 3 days earlier with low dose L-PAM (
), anti-B7-2 (