Importance of the B7-2 Molecule for Low Dose Melphalan-Induced Acquisition of Tumor-Eradicating Immunity by Mice Bearing a Large MOPC-315 Tumor

Margalit B. Mokyr, Tatiana V. Kalinichenko, Leonid Gorelik and Jeffrey A. Bluestone
J Immunol February 15, 1998, 160 (4) 1866-1874;

Abstract

We have previously shown that low dose melphalan (l-phenylalanine mustard; l-PAM) therapy of hitherto immunosuppressed mice bearing a large (20-mm) s.c. MOPC-315 tumor leads to the acquisition of potent CD8+ T cell-mediated antitumor immunity which in turn eradicates the large tumor burden not eradicated by the direct antitumor effects of the drug. Here we show the preferential importance of the B7-2 costimulatory molecule for the curative effectiveness of low dose l-PAM for mice bearing a large MOPC-315 tumor by demonstrating that treatment with anti-B7-2 mAb, but not anti-B7-1 mAb, reduced the percentage of mice cured by the low dose l-PAM. In addition, we show the preferential importance of the B7-2 molecule for the low dose l-PAM-induced acquisition of the ability of tumor-infiltrating lymphocytes from MOPC-315 tumor bearers to secrete IL-2 and IFN-γ 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.

T cell activation requires two distinct signals. The first signal is Ag specific and originates from the interaction of TCR with Ag presented on APC in the context of MHC. The second signal is provided either by soluble factors such as IL-2 or by the interaction of costimulatory receptors and their ligands. One of the most important costimulatory receptors on T cells appears to be the CD28, given that blockade of CD28 signaling for T cell activation can cause clonal anergy or deletion (1, 2).

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-γ 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.

Materials and Methods

Tumors

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 × 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 × 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-γ 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 × 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 × 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-γ. 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-γ 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-γ ELISA, we used rat anti-mouse IFN-γ mAb R46A2 (American Type Culture Collection, Rockville, MD) as capture Ab and biotinylated rat anti-mouse IFN-γ mAb XMG1.2 (PharMingen) as detection Ab. The sensitivity of the ELISA for IL-2 is 15 pg/ml, and that for IFN-γ 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)] × 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 × 107 effector cells, wherein 1 LU is defined as the number of effector cells producing 20% lysis of 1 × 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.

Results

Effect of anti-B7-1 or anti-B7-2 mAb treatment on the curative effectiveness of low dose l-PAM for mice bearing a large MOPC-315 tumor

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.

FIGURE 1.
FIGURE 1.

Effect of anti-B7-1 (•) or anti-B7-2 (▴) 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 ≤ 0.05) reduction in the survival time relative to mice treated with l-PAM plus NIgG.

FIGURE 2.
FIGURE 2.

Effect of anti-B7-1 mAb on the rate of tumor regression in mice bearing a large MOPC-315 tumor and treated with low dose l-PAM. Mice bearing a 20-mm s.c. MOPC-315 tumor received 1.75 mg/kg l-PAM in conjunction with either anti-B7-1 mAb (•) or NIgG (▪). Numbers in parentheses indicate the number of mice in the subgroup per total number of mice subjected to the treatment protocol. * indicates statistically significant (p ≤ 0.05) larger tumors relative to mice treated with l-PAM plus NIgG.

Effect of low dose l-PAM therapy on B7-1 and B7-2 expression in the tumor nodules of MOPC-315 tumor bearers

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.

FIGURE 3.
FIGURE 3.

Flow cytometric analysis of B7-1 and B7-2 expression on small cells (left two histograms) or large cells (right two histograms) among cells derived from the tumor nodules of untreated or low dose l-PAM-treated MOPC-315 tumor bearers. Cells derived from the s.c. tumor nodules of untreated mice (light solid line) or mice treated 2 (heavy dotted line) or 3 (heavy solid line) days earlier with low dose l-PAM (2.5 mg/kg) were stained with biotinylated anti-B7-1 mAb (top histograms), biotinylated anti-B7-2 mAb (bottom histograms), or biotinylated NIgG (light dotted line; in all histograms), followed by PE-conjugated streptavidin. Gating on the small or large cells was done on the basis of light scatter properties.

FIGURE 4.
FIGURE 4.

Two-color flow cytometric analysis of B7-1 and B7-2 expression on B220+, MAC-1+ or CD3+ cells among small cells (left two histograms) and among large cells (right two histograms) from the tumor nodules of untreated mice (□) and mice treated 3 days earlier with low dose l-PAM (▪). Data are plotted as fold increase in MFI which represents the fold increase in the MFI of a given cell subset exposed to biotinylated anti-B7-1 or anti-B7-2 mAb plus PE-conjugated streptavidin over that of the same cell subset exposed to biotinylated NIgG plus PE-conjugated streptavidin.

Effect of low dose l-PAM therapy on B7-1 and B7-2 expression in the draining lymph nodes of MOPC-315 tumor bearers

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.

FIGURE 5.
FIGURE 5.

Flow cytometric analysis of B7-1 and B7-2 expression on cells derived from the draining lymph nodes of untreated or low dose l-PAM-treated MOPC-315 tumor-bearing mice. Lymph node cells derived from untreated tumor-bearing mice (light solid line) or mice treated 2 (heavy dotted line) or 3 (heavy solid line) days earlier with low dose l-PAM (2.5 mg/kg) were stained with biotinylated anti-B7-1 mAb (top histogram), biotinylated anti-B7-2 mAb (bottom histogram), or biotinylated NIgG (light dotted line in both histograms), followed by PE-conjugated streptavidin.

Studies were conducted next to identify the cell type(s) that display up-regulation of B7-2 expression in the draining lymph nodes of low dose l-PAM-treated MOPC-315 tumor bearers. Specifically, we studied the expression of the B7-2 molecule on B220+, MAC-1+, or CD3+ cells from the draining lymph nodes of untreated tumor-bearing mice and of tumor-bearing mice treated 3 days earlier with low dose l-PAM. As seen in Figure 6⇓, 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.

FIGURE 6.
FIGURE 6.

Two-color flow cytometric analysis of B7-2 expression on B220+, MAC-1+, or CD3+ cells among cells derived from the draining lymph nodes of untreated tumor-bearing mice (□) and tumor-bearing mice treated 3 days earlier with low dose l-PAM (▪). Data are plotted as fold increase in MFI which represents the fold increase in the MFI of a given cell subset exposed to biotinylated anti-B7-1 or anti-B7-2 mAb plus PE-conjugated streptavidin over that of the same cell subset exposed to biotinylated NIgG plus PE-conjugated streptavidin.

Effect of anti-B7-1 or anti-B7-2 mAb treatment on IL-2 and IFN-γ 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.

FIGURE 7.
FIGURE 7.

Effect of anti-B7-1 or anti-B7-2 mAb treatment on the level of IL-2 (top) or IFN-γ (bottom) produced by TILs from low dose l-PAM-treated MOPC-315 tumor bearers. TILs derived from MOPC-315 tumor bearers treated with low dose l-PAM (2.0 mg/kg) plus anti-B7-1, anti-B7-2, or NIgG were cultured for 24 h. Subsequently, the culture supernatants were assessed for the level of IL-2 or IFN-γ produced through the use of an ELISA. Data are presented as the mean ± SE for cytokine production from five different experiments. * indicates a statistically significant (p ≤ 0.05) decrease relative to the level of cytokine produced by TILs from mice treated with l-PAM plus NIgG.

Next, we determined the effect of anti-B7-1 or anti-B7-2 mAb treatment on low dose l-PAM-induced up-regulation of IFN-γ 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-γ production at the tumor site (33, 34) and 2) neutralization of IFN-γ 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-γ 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-γ 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-γ 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-γ 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-γ 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-γ than TILs from mice treated with l-PAM plus NIgG. In fact, the magnitude of reduction in the level of IFN-γ 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-γ.

FIGURE 8.
FIGURE 8.

Effect of anti-B7-1 or anti-B7-2 mAb treatment on the overall potential of TILs from low dose l-PAM-treated MOPC-315 tumor bearers to produce IFN-γ in response to stimulation with MOPC-315 tumor cells. TILs derived from MOPC-315 tumor bearers treated with low dose l-PAM (2.0 mg/kg) plus anti-B7-1, anti-B7-2, or NIgG were cultured for 24 h in the presence (▪) or the absence (□) of 10 ng/ml PMA. Subsequently, the culture supernatants were assessed for the level of IFN-γ produced through the use of an ELISA. Data are presented as the mean ± SE for IFN-γ production from three different experiments. *indicates a statistically significant (p ≤ 0.05) increase relative to the level of IFN-γ produced by the same TILs cultured in the absence of PMA. ** indicates a statistically significant (p ≤ 0.05) decrease relative to the level of IFN-γ produced by the TILs from mice treated with l-PAM plus NIgG when cultured under the same conditions.

Effect of anti-B7-1 or anti-B7-2 mAb treatment on the anti-MOPC-315 CTL activity exhibited by TILs from low dose l-PAM-treated MOPC-315 tumor bearers

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.

FIGURE 9.
FIGURE 9.

Effect of anti-B7-1 or anti-B7-2 mAb treatment on the level of anti-MOPC-315 lytic activity of TILs from low dose l-PAM-treated MOPC-315 tumor bearers. TILs derived from individual MOPC-315 tumor bearers treated with low dose l-PAM plus anti-B7-1 (▨), anti-B7-2 (□) or NIgG (▪) were evaluated for their lytic activity by the 3.5-h 51Cr release assay. Data are presented as the mean ± SE for TILs from 12, 9, or 10 individual mice treated with l-PAM plus anti-B7-1, anti-B7-2, or NIgG, respectively. * indicates a statistically significant (p ≤ 0.05) decrease relative to the lytic activity exhibited by TILs from mice treated with l-PAM plus NIgG.

Discussion

Several studies in nontumor systems have suggested that the endogenously expressed B7-1 and B7-2 molecules are not simply interchangeable ligands in the same costimulatory pathway (2, 11, 12, 13). For example, Kuchroo et al. (12) have shown that anti-B7-1 mAb reduces the incidence of experimental allergic encephalomyelitis (which is mediated by type 1 cytokines) through enhancement in IL-4 production, while anti-B7-2 mAb treatment increases the severity of the disease through enhancement in IFN-γ 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-γ 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-γ 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-γ (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-γ 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.

Footnotes

  • ↵1 Supported by Research Grant IM-435 from the American Cancer Society.

  • ↵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 × 104 target cells; PE, phycoerythrin.

  • Received March 13, 1997.
  • Accepted October 29, 1997.

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The Journal of Immunology
Vol. 160, Issue 4
15 Feb 1998
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