Tumoricidal Effects of Activated Macrophages in a Mouse Model of Chronic Lymphocytic Leukemia

Abstract

The Eμ-TCL1 transgenic mouse spontaneously develops a CD5⁺ B cell lymphoproliferative disorder similar to human chronic lymphocytic leukemia (CLL). Given the ineffectual T cell antitumor responses in this mouse model of CLL, we sought to determine whether combined treatment with anti-CD40 mAb (αCD40) and CpG-containing oligodeoxynucleotides (CpG) could exert immunotherapeutic effects. We have previously shown that macrophages activated by sequential ligation of CD40 and TLR9 could become cytotoxic against solid tumor cell lines both in vitro and in vivo. In the current study, we find that αCD40 plus CpG-activated macrophages induce tumor B cell apoptosis in vitro and that αCD40 plus CpG treatment markedly retards tumor growth in immunodeficient SCID/Beige mice following transplantation of primary tumor B cells. Our results suggest a novel immunotherapeutic strategy for CLL that may be effective even in the face of tumor or chemotherapy-induced T cell immunodeficiency.

Chronic lymphocytic leukemia (CLL)⁴ is characterized by the progressive accumulation of CD5⁺ B cells with a low mitotic rate but prolonged cell survival. Despite the fact that the tumor B cells express high levels of class I and II MHC and a tumor-specific Ag (their unique Ig protein), CLL cells are poor APC, due in part to their production of inhibitors of T cell activation such as TGF-β and soluble CD27 (1, 2). Stimulation of the CD40 pathway enhances the immunogenicity of CLL cells by up-regulating adhesion and costimulatory molecules, such as CD80, CD86, and CD70 (3, 4), provides a promising immunotherapy approach. Although this approach is efficacious in vitro (and in vivo in some mouse lymphoma models; Ref. 5-7), it remains unclear whether it can reverse the profound T cell dysfunction (8, 9) in vivo in human patients to the extent necessary for an effective T cell dependent antitumor immune response.

Immunostimulatory class B CpG also have been used in experimental tumor immunotherapy alone or as an adjuvant to other stimulants. CpG up-regulates the expression of surface CD40, CD80, CD86, and MHC class I on human CLL B cells (10). Costimulation with CpG and CD40-ligand transfected mouse fibro-blasts results in a synergistic up-regulation of costimulatory molecules, enhancing APC function in an allogeneic MLR (10). Most of these approaches focus on inducing T cell-mediated antitumor effects by augmenting the APC function of tumor cells. However, patients with CLL frequently exhibit some level of T cell immunodeficiency due to tumor-derived factors (1, 2) as well as chemotherapy (11, 12). Therefore, T cell-independent antitumor strategies in CLL warrant investigation as an alternative immunotherapeutic approach, either alone or as an adjuvant to conventional or to T cell-dependent immune based therapies.

In this study, we use a mouse model of CLL, the Eμ-TCL1 transgenic mouse developed in the laboratory of C. Croce (13). This mouse, which overexpresses the TCL1 (T cell lymphoma/leukemia-1) oncogene in B cells, spontaneously develops a hyperplasia of CD5⁺ B cells in the peripheral blood and peritoneum, which progresses toward a monoclonal B cell leukemia/lymphoma with infiltration of spleen, bone marrow, and other organs. The histological and phenotypic features of the tumor B cells in the Eμ-TCL1 mouse mimic those in human CLL. In the intact Eμ-TCL1 mouse, there is an ineffectual T cell antitumor response despite, as we report in this study, the high, constitutive tumor cell expression of CD80 and CD86. This compelled us to examine the utility of activated macrophages (Mϕ) as a T cell-independent immunotherapeutic tool in this mouse model of CLL.

We have previously shown that Mϕ activated by CD40 ligation alone or in combination with CpG, are cytotoxic against CD40 negative solid tumor cell lines (14, 15). In this report, we describe antitumor responses generated by anti-CD40 (αCD40) plus CpG activated Mϕ (αCD40 plus CpG-Mϕ) against this spontaneous, primary model of CLL in which the tumor cells also express receptors for CD40L and CpG, suggesting a novel immunotherapeutic strategy for CLL that may be effective even in the face of T cell inhibition by chemotherapy or tumor-derived factors.

Materials and Methods

Mice and cell lines

C57BL/6, BALB/c, SCID/Beige mice (Harlan Sprague Dawley), and Eμ-TCL1 transgenic in C3H/B6 background (provided by Dr. Carlo Croce, The Ohio State University, Ohio) were housed, cared for, and used in accordance with the Guide for Care and Use of Laboratory Animals under a protocol approved by the University of Wisconsin Animal Care and Use Committee. The A20 B cell lymphoma line was grown in RPMI 1640 plus 10% FCS. CD40L or control CD32 (Ig Fc receptor) transformed L cells were cultured in DMEM plus 10% FCS, and 0.5 μM 2-ME.

Abs and reagents

The FGK 45.5 hybridoma producing αCD40 was a gift from Dr. F. Melchers (Basel Institute, Switzerland). αCD40 was produced as previously described (15). Endotoxin-free CpG1826 (TCCATGACGTTCCT GACGTT; CpG motifs that are absent in control non-CpG1982 are bold and underlined) was purchased from Coley Pharmaceuticals Group. Antimouse CD19 allophycocyanin, CD5-PE, CD80-FITC, CD86-PE, CD70-Biotin, CD4-Biotin, CD8-Biotin, F4/80 allophycocyanin, and purified anti-Fas-ligand mAbs were purchased from eBioscience. CD40-FITC and Annexin-5-FITC mAbs were purchased from BD Pharmingen. LPS (Escherichia coli 0111:B4LPS) was purchased from InvivoGen.

Activation of Mϕ with αCD40 and CpG1826

Mice were injected i.p. with 0.5 mg of either αCD40 or control rat IgG (Sigma-Aldrich) in 0.5 ml PBS. Three days later, unless otherwise specified, peritoneal cells were harvested and enriched for Mϕ by adhesion to plastic (15). Mϕ-enriched adherent cells (>98% F4/80⁺ by flow cytometric analysis) were incubated in vitro for 24 h in medium with or without 5 μg/ml CpG1826 or 1 μg/ml LPS. In preliminary experiments, no difference in Mϕ stimulatory capacity of control non-CpG1982 and PBS was observed; therefore, PBS was used as a control for CpG1826 in all subsequent experiments.

[³H]Thymidine ([³H]TdR) incorporation assay

Splenocytes from an Eμ-TCL1 tumor-bearing mouse (70% CD19⁺CD5⁺ tumor B cells) were incubated with 25 μg/ml αCD40 or control rat IgG. Medium or 1 μg/ml CpG were added 72 h later and incubated for additional 24 h. For the last 6 h, wells were pulsed with 1 μCi/well of [³H]TdR. [³H]TdR incorporation was quantified using a beta counter (MicroBeta Trilux, PerkinElmer Life Sciences).

Adhesion-purified Mϕ (1.5 × 10⁵ per well) from mice treated with αCD40 or control rat IgG were incubated with tumor cells (1 × 10⁵ per well) for 24 h in medium with or without 1 μg/ml LPS or 5 μg/ml CpG. In some experiments, 5 μg/ml anti-mouse FasL (14-5911, eBioscience), or 5 mM l-nitro-arginine-methyl-esterase (l-NAME; Sigma-Aldrich) were added in the coculture. For the last 6 h, wells were pulsed with 1 μCi/well of [³H]TdR, and incorporation was quantified as previously described (15). Results are presented as either counts per 5 min or percentage of inhibition of [³H]TdR incorporation into tumor cells, calculated according to the formula ([a - b]/a) × 100%, where a is [³H]TdR-counts from tumor cells cultured in medium with or without CpG and blocking reagents and in the absence of Mϕ, and b is [³H]TdR counts from tumor cells cultured in the presence of Mϕ and additional indicated reagents.

Flow cytometry

Cell suspensions were stained with 1 μg of indicated Abs for 30 min at 4°C in PBS plus 2% FCS. To avoid nonspecific binding of Mϕ, total peritoneal cells were incubated with blocking rat IgG for 20 min at 4°C before staining. Cytometer analysis was done using an LSRII or FACSCalibur (BD Pharmingen). The data were analyzed using FlowJo (TreeStar) or CellQuest (BD Pharmingen) software.

Mixed lymphocytic reaction

CD40L-L cells and CD32-L cells (5 × 10⁶ cells/ml) were treated with 100 μg/ml mitomycin-C (Sigma-Aldrich) for 1 h at 37°C and washed using medium. Splenocytes from an Eμ-TCL1 tumor-bearing mouse (C3H/B6 background, 68-90% CD19⁺CD5⁺ tumor B cells) were cultured with medium or mitomycin-C pretreated CD40L-L cells or CD32-L cells at a 2:1 ratio, or with 50 μg/ml αCD40 for 48 h. The nonadherent Eμ-TCL1 splenocytes were collected, washed, and irradiated by a Cs source (3000 R) or incubated with 100 μg/ml mitomycin-C followed by six washes, then incubated at increasing ratios with 7.5-10 × 10⁴ anti-CD4 and anti-CD8 MACS bead (Miltenyi Biotec) positively selected BALB/c or C57/Bl6 spleen T cells (the purity was 80-90% by flow cytometry analysis) and cultured for 120 h. [³H]TdR uptake was measured for the final 8-16 h of culture. Results are presented as the proliferation index (the ratio of average cpm for triplicate wells in the presence of stimulator cells to background T cell proliferation).

Apoptosis assay

Splenocytes from an Eμ-TCL1 tumor-bearing mouse were incubated with 25 μg/ml rat IgG or αCD40 with or without 1 μg/ml CpG. Cells were collected 24-72 h later and flow cytometric analysis of apoptotic changes was performed by using 3,3′-dihexiloxa dicarbocyanine (DiOC₆), propidium iodide (PI) (both from Invitrogen), anti-Annexin-5-FITC staining as readouts for apoptotic and dead cells as previously described (16).

Adhesion-purified Mϕ (1.5 × 10⁵ per well) from mice treated with αCD40 or control rat IgG were incubated with tumor cells (1 × 10⁵ per well) for 24 h in medium with or without 5 μg/ml CpG. Twenty-four hours later, the tumor cells were assessed for expression of phosphatidylserine by staining with Annexin V-FITC as a measure of early apoptotic changes, and tested for changes in tumor cell membrane integrity by staining with DNA-binding dye 7-aminoactinomycin D (BD Pharmingen) as a measure of late apoptotic changes (17).

NO detection

Rat IgG- or αCD40-Mϕ were cocultured for 24 h in vitro with splenocytes from Eμ-TCL1 tumor bearing mice in medium with or without 5 μg/ml CpG in the presence or absence of 5 μg/ml anti-mouse FasL or 5 mM l-NAME. Nitrite accumulation in cell culture supernatants was determined using the Griess reagent (Sigma-Aldrich) as described (15).

In vivo tumor model

Eμ-TCL1 tumor-bearing mice were depleted of T cells by i.p. injection of a mixture of 0.5 mg of anti-CD4 mAb (GK1.5) and 0.5 mg of anti-CD8 mAb (2.43) 5 days before spleen harvest. On day 0, a total of 1 × 10⁶ splenocytes from T cell depleted, tumor-bearing Eμ-TCL1 donor mice were engrafted i.p. into SCID/Beige mice. In the preliminary and first full experiments αCD40 and CpG were injected on a weekly schedule. In the experiment detailed in Fig. 5, the recipients received 0.25 mg of αCD40 i.p. on day 5, 19, 33, 47, and 61 after tumor implantation and 0.025 mg of CpG on day 8, 22, 36, 50, and 64. Control mice received 0.25 mg of rat IgG and 1 ml of PBS, respectively. Antitumor effects were evaluated on day 14, 28, 42, 56, and 70 after tumor implantation, at which point control mice began to be euthanized due to moribund status. Tumor load was approximated by the percentage of tumor cells in the peripheral blood, which we found to correlate well with total tumor load in spleen and peritoneum (Q.W. and E.A.R., unpublished observations).

FIGURE 5.

αCD40 plus CpG treatment is effective in vivo in the absence of T cells. A, Groups of eight SCID/Beige mice were inoculated i.p. with 1 × 10⁶ splenocytes from T cell depleted Eμ-TCL1 tumor-bearing donor mice and then treated with 0.25 mg of αCD40 (white) or rat IgG (black) by i.p. injection, followed 3 days later by i.p. injection of 0.025 mg of CpG (white) or PBS (black). This was begun on day 5 post transplant and continued every 2 wk for five cycles as noted by arrows in A. Tumor development was monitored by flow cytometric analysis of peripheral blood from the tail vein at 2 wk intervals until mice in both groups needed to be euthanized due to moribund status. Tumor burden is expressed as percentage of CD19⁺CD5⁺ tumor cells in the peripheral blood lymphocytes. Points, mean; bars, SE; *, p < 0.05; when compared with control group (n = 8) by Student’s t test. The experiments are representative of one pilot and three full independent experiments. B, Tumor related survival of these populations is shown and are statistically significantly different at p < 0.02 by log-rank test.

Results

CD40 stimulation enhances APC function of tumor B cells

CD40 ligation induces the expression of costimulatory B7 family molecules on human CLL tumor B cells, increasing their APC function (3). In contrast to human CLL, the CD5⁺ tumor cells in the Eμ-TCL1 mice constitutively express surprisingly high levels of B7-1 (CD80) and B7-2 (CD86) (Fig. 1A). Given that B7 molecule up-regulation is usually considered a major factor in enhanced APC function of B cells, we assessed whether the APC function of Eμ-TCL1 tumor B cells still could be further enhanced by CD40 stimulation. Of note, CD40 stimulation does increase the expression of another important costimulatory molecule, CD70, on the tumor B cells, but does not significantly enhance the already high levels of CD80 (data not shown) and CD86 expression (Fig. 1B). We have previously shown that CD70 up-regulation augments APC function in human normal and CLL B cells (2). CD40 ligation, either via recombinant CD40L or anti-CD40 mAbs, enhances the Ag-presenting immunogenicity of mouse tumor B cells in an MLR, but the increased level of T cell proliferation is paradoxically diminished when larger numbers of tumor cells are used as stimulator cells (Fig. 1C). These results suggest that while CD40 cross-linking can augment the costimulatory capacity of tumor B cells, this effect may be mitigated by tumor-mediated suppression of T cell responses. The mechanisms of this inhibition are unknown and are under investigation, but suggest that augmentation of tumor cell killing by T cell-independent means may be efficacious in this CLL model. We previously have shown that activated Mϕ were cytotoxic against melanoma and neuroblastoma tumorcell lines in vitro and in vivo (14), and aimed to evaluate their potential in CLL where T cell immunodeficiency is well documented.

FIGURE 1.

CD40 stimulation enhances APC function of tumor B cells without altering CD80/CD86 levels. Total spleen cells from an Eμ-TCL1 tumor-bearing mouse were stained with anti-CD19, CD5, and CD40, CD80, or CD86 freshly after isolation (A) and following 24 h of culture with αCD40 or control IgG (B). Tumor B cells (CD5⁺) were compared with T cells and residual normal B cells (CD5^-). C, Spleen cells from an Eμ-TCL1 tumor-bearing mouse were precultured with medium (black) or αCD40 (gray) for 48 h. Mitomycin-treated tumor cells incubated at different cell ratios with 7.5 × 10⁴ MACS bead positively selected C57BL/6 T cells. [³H]TdR uptake was measured for the final 8 h of a 120 h culture. Results are presented as the proliferation index (the ratio of average counts per min for triplicate wells in the presence of stimulator cells to background T cell proliferation) in one representative experiment of six performed with both αCD40 and CD40L-expressing L cells (data not shown). *, p < 0.01 αCD40 vs medium control.

αCD40 stimulates Eμ-TCL1 tumor B cells to proliferate and protects them from apoptosis

As we wished to use CD40 cross-linking and TLR costimulation to activate Mϕ effector function in vitro and in vivo, we first sought to determine whether αCD40 and CpG caused any direct cytotoxic effects on these CD40⁺ tumor B cells. CD40 ligation of some low-grade B-lymphoma cells, such as human CLL B cells, promotes cell survival and proliferation, though it also may sensitize the cells to apoptotic signals delivered later (18). In Eμ-TCL1 tumor B cells, αCD40 treatment increases the proliferation of both CD5⁺ tumor B cells (Fig. 2A) and CD5^- normal B cells (data not shown) compared with control rat-IgG treatment. αCD40 itself does not cause apoptosis of tumor B cells, but rather provides modest protection from apoptosis, based on both annexin V and DiOC₆, and PI staining as readouts for apoptotic and dead cells, respectively, after 24 (data not shown) or 72 h (Fig. 2B), when compared with the control rat IgG treatment. CpG also provides a moderate degree of protection from apoptotic death for tumor B cells (Fig. 2B). These results suggest that, separately, αCD40 and CpG treatments have relatively mild prosurvival and moderate mitogenic effects on Eμ-TCL1 tumor B cells, similar to that noted in human CLL cells. Together, however, αCD40 and CpG have synergistic effects on Eμ-TCL1 tumor B cell proliferation and survival (Fig. 2). Thus, any antitumor effects of activated Mϕ must overcome the proliferative and anti-apoptotic effects of these stimuli.

FIGURE 2.

αCD40 and CpG stimulate Eμ-TCL1 tumor B cell proliferation and do not directly induce apoptosis. A, Spleen cells from an Eμ-TCL1 tumor-bearing mouse (70% CD19⁺CD5⁺ tumor B cells) were incubated with 25 μg/ml rat IgG or αCD40. Seventy-two hours later, medium (10 μl) or 1 μg/ml CpG were added and incubated for an additional 24 h. Proliferation was measured by [³H]TdR incorporation assay, as described in Materials and Methods. Results are presented as counts per 1 min for triplicate wells ± SEM. *, p < 0.05; **, p < 0.01 when compared with rat IgG group. B, Spleen cells from an Eμ-TCL1 tumor-bearing mice (90% CD19⁺CD5⁺ tumor B cells) were incubated with αCD40 or control rat IgG (25 μg/ml) for 72 h. At this point, medium or CpG (1 μg/ml) was added. Twenty-four hours later, flow cytometric analysis of apoptotic changes of the tumor cells was performed using DiOC₆ (abscissa), antiannexin V mAb (ordinate, right column), and PI (ordinate left column). Low DiOC₆ staining accompanied by exclusion of PI indicates early mitochondrial changes associated with apoptosis (cells in the left lower quadrant in left column). When these PI negative cells are gated, DiOC6 low cells express higher levels of surface annexin V, an alternative marker of apoptosis (right column). Numbers shown reflect percentage of cells in each quadrant or gate. Results are representative of two separate experiments.

αCD40 plus CpG-Mϕ exhibit antitumor effects in vitro

We previously have shown that Mϕ activated by αCD40 plus CpG were cytotoxic against tumor cell lines that lack CD40 expression (14). In this study, we ask whether such αCD40 plus CpG-Mϕ could inhibit CD40 expressing, spontaneously arising tumor cells in vitro and in vivo. Control rat IgG-treated Mϕ alone inhibit the proliferation of A20 cells (mouse lymphoma line, Fig. 3A), but mildly augment proliferation of primary Eμ-TCL1 tumor cells (Fig. 3B) in vitro, either in the presence or absence of TLR stimuli CpG or LPS (data not shown). Indeed, Mϕ pretreated with rat IgG also fail to produce NO in the presence of tumor targets (Fig. 4, A and B). In contrast, preactivated αCD40-Mϕ strongly inhibit proliferation of tumor B cells in the absence of TLR stimulation and virtually eliminate growth after stimulation with CpG (Figs. 3B and 4,C and D) or LPS (data not shown). This is mirrored by a significant release of NO by Mϕ (Fig. 4A). As expected, tumor B cells alone do not show production of NO, even in the presence of CpG (data not shown).

FIGURE 3.

Activated Mϕ can inhibit growth and survival of B cell tumors in vitro. Peritoneal Mϕ from rat IgG- and αCD40-treated C57BL/6 mice (1.5 × 10⁵ per well) were incubated with A20 cells (A) or primary Eμ-TCL1 tumor cells (B)(1 × 10⁵ per well) for 24 h in medium with (gray) or without (black) 5 μg/ml CpG. For the last 6 h, wells were pulsed with 1 μCi/well of [³H]TdR. Results are presented counts per 5 min for triplicate wells ± SE of [³H]TdR uptake into the tumor cells and are representative of four independent experiments using either CpG or LPS as a secondary stimulus, which gave similar results. *, counts <1000. A20 (C) or primary Eμ-TCL1 tumor cells (D) were cultured in medium only, rat IgG, or αCD40-actived Mϕ in the presence (right) or absence (left) of 5 μg/ml CpG. Twenty-four hours later, the tumor cells were assessed for expression of phosphatidylserine by staining with Annexin V-FITC, and assessed for tumor cell membrane integrity by staining with 7-aminoactinomycin D. Numbers shown reflect percentage of cells in each quadrant.

FIGURE 4.

NO but not Fas-FasL is involved in antitumor effect of αCD40 plus CpG-Mϕ.Mϕ from rat IgG-or αCD40-treated young (2 mo old) C57BL/6 (A and C) or aged (10-12 mo) Eμ-TCL1^-/- mice (B and D) were tested in vitro for antitumor activity against Eμ-TCL-1 tumor B cells or A20 mouse B-lymphoma cells in [³H]TdR incorporation assay, as described in Materials and Methods and Fig. 3, A and B, in medium with or without 5 μg/ml CpG. Anti-FasL (5 μg/ml) or l-NAME (5 mM) was added in the culture at initiation of the experiment. Results are presented as nitrite level in culture supernatants (A and B) and percentage of inhibition of tumor cell proliferation (C and D), calculated as described in Materials and Methods. Results are presented as means ± SE for triplicate wells. Data representative of three independent experiments are shown. *, not detectable.

The tumoristasis mediated by αCD40 plus CpG-Mϕ is associated with the induction of apoptosis in the tumor cells. Unlike rat IgG-conditioned Mϕ, αCD40-Mϕ strongly induce apoptosis in both the A20 (Fig. 3C) and Eμ-TCL1 tumor cells (Fig. 3D) upon in vitro coculture when stimulated with CpG (Fig. 3, C and D) or LPS (data not shown).

Mϕ possess a number of potentially cytotoxic soluble and cell surface mediators for the killing Eμ-TCL1 tumor B cells. It is known that CD40-cross-linking induces Fas (CD95) expression on B cells (18, 19), which could potentially prime the CLL B cells to be killed by FasL-expressing immune effectors including activated Mϕ. Indeed, after in vivo activation by CpG, peritoneal Mϕ substantially up-regulate expression of FasL (CD178) (data not shown). Fas expression on tumor B cells also is increased after in vivo treatment, particularly with CpG. At least in vitro, however, blocking FasL does not affect the inhibition of tumor cell proliferation (Fig. 4, C and D) or the induction of tumor cell apoptosis (data not shown) by αCD40 plus CpG-Mϕ, suggesting that Fas-FasL interaction may not be a critical mechanism of Mϕ-inducedtumor cell death. This result is in agreement with the observation that CpG activation alone, while strongly up-regulating Mϕ Fas-L and B cell Fas expression, does not result in substantial Mϕ-induced killing (Fig. 3). As we have described previously (14), and confirm in this study, the tumoricidal capacity of activated Mϕ is elicited by stimuli that generate significant NO production (Fig. 4). Consistent with these findings, l-NAME, an inhibitor of NO synthase, substantially reduces the ability of activated Mϕ to kill tumor B cells in vitro (Fig. 4, C and D), demonstrating that NO is important in this process.

One appealing aspect of Mϕ-based immunotherapy is its limited dependence on T cell function. This may be critical in chemotherapy-treated and/or elderly patients whose T cell numbers and/or function are depressed. Eμ-TCL1 mice are at least 4 mo old when the oligoclonal “preleukemia” can be observed in the peripheral blood and usually are more than 10 mo old when they have >80% tumor cells in the peripheral blood and spleen. We tested whether αCD40-Mϕ from aged (10-mo-old) Eμ-TCL1 transgene-negative littermate mice could suppress the B cell leukemia cells as well as Mϕ from young animals. αCD40-Mϕ or rat IgG-Mϕ from aged mice (Fig. 4D) suppressed primary tumor B cells to the same extent as Mϕ from the originally used young C57BL/6 wild-typemice (Fig. 4C), though with lower NO production by the Mϕ from older mice (Fig. 4, B vs A). Thus, while NO seems to be involved in Mϕ-mediated killing, there may be a necessary minimum threshold rather than a straight dose-response relationship.

αCD40 plus CpG treatment induces antitumor effect in vivo

We previously have found that αCD40 and CpG synergize in vivo in inhibiting B16 melanoma cell line growth when CpG is administered 3 days following αCD40, as CD40 stimulation up-regulates TLR-9 expression on Mϕ, thereby “priming” them to receive CpG-mediated activation signals (14). We therefore used this schedule for the in vivo experiments, with αCD40 plus CpG administered i.p. every 1-2 wk.

We found that the injection of 10⁶ primary tumor cells consistently results in the outgrowth of a phenotypically identical B cell lymphoma in SCID/Beige recipients. The transplanted tumor can be found in the hosts’ peripheral blood 4 wk after i.p. injection of anti-CD4 and anti-CD8 T cell-depleted, tumor-bearing Eμ-TCL1 mouse splenocytes (Fig. 5A). Peripheral blood tumor burden is an accurate indicator of tumor cell percentages in the spleen and peritoneum, both in intact Eμ-TCL1 mice and in SCID/Beige transplant recipients (data not shown). SCID/Beige hosts were treated with five cycles of αCD40 plus CpG, every 2 wk, beginning 5 days posttransplant. Treatment was halted when control mice exhibited high tumor burden and required euthanasia. Even with this limited course of therapy, αCD40 plus CpG induced significant in vivo antitumor effects in SCID/Beige mice that resulted in retardation of tumor growth (Fig. 5A) and a 63% increase in tumor-related survival (Fig. 5B, mean tumor-related survival 130 days in αCD40/CpG group (95% confidence interval 120-140) and 80 days in rIgG/PBS group (95% confidence interval 70-100)). By Cox proportional hazard model analysis, the relative risk of death in control vs αCD40/CpG-treated mice was 3.61 (95% CI of 1.01-12.94, p = 0.049). In four separate experiments, no SCID/Beige recipient was “cured” of tumor from αCD40 plus CpG treatment. In these immunocompromised mice, the αCD40/CpG was fairly toxic, resulting in death of up to 25% of SCID-beige animals before tumor emergence. Previous experience with similar regimens in immunocompetent C57BL/6 and A/J mice did not show this level of toxicity (I.N.B., unpublished observations). The differential toxicity may be related to the lack of IL-10 production in SCID Mϕ following CpG stimulation (20), leading to unopposed toxicity of proinflammatory cytokines.

Discussion

The use of stimulatory αCD40 Abs in tumor immunotherapy has been successful against transplantable tumor cell lines, including CD40 negative solid tumor lines (14, 15) and CD40 positive solid tumor and B cell lymphoma lines (6, 7, 21, 22), but has shown limited efficacy against CD40 negative lymphoma lines (7). Thevast majority of these studies have focused on the induction of T cell-mediated antitumor responses. They have shown that CD40-induced activation of tumor cells and/or professional APC such as dendritic cells results in CD8⁺ T cell dependent tumor killing and immunologic memory. This may be mediated through direct activation of CD8⁺ T cells by CD40-stimulated tumor or APC cross-presenting tumor Ags in a CD4-independent fashion (7), but also appears to circumvent CD4⁺ T cell tolerance in other models (22). In lymphoma (cell line) models of immunotherapy with CD40 ligation, a direct role for activated Mϕ has not been described. French et al. (7) found that SCID mice transplanted with CD40⁺ BCL1 lines were not protected by αCD40 treatment unless BALB/c splenocytes also were transferred, suggesting the need for lymphoid cells. The dosing of αCD40 Ab was only over a 3-day period, however, and did not contain TLR agonists, in contrast to the current study. Interestingly, these authors found that contrary to most successful tumor immunotherapy protocols, treatment shortly after tumor cell inoculation, or transferring fewer tumor cells, was far less effective than treatment given later or after inoculation with increased numbers of tumor cells (6, 7). Nonetheless, early (day 2-5) treatment did prolong survival by 10-15 days, suggesting some antitumor effect that could be consistent with a Mϕ response, which, in the model of French et al. (6, 7), impeded the development of a curative T cell response (presumably due to a shortage of target cells needed for T cell activation during αCD40 treatment).

The current study, using a primary murine tumor model that accurately simulates human CLL, indicates that Mϕ activated through CD40 and TLR are capable of killing primary, spontaneously arising lymphoma B cells in vivo and in vitro. The exact mechanism of tumor B cell inhibition and killing by αCD40 plus CpG-Mϕ is not clear, but it appears dependent on production of NO. Upon activation, Mϕ can express a number of factors, such as TNF-α, NO, IFN-γ, IFN-α, IL-1, TRAIL, and FasL. Despite upregulation of FasL on Mϕ and Fas on target B cells in vivo following αCD40 plus CpG-treatment, our data suggests that Fasmediated B cell killing is unlikely to be a critical mechanism in this system. This is consistent with our observation that αCD40 and LPS-activated Mϕ from mice lacking functional FasL are able to kill CD40^- tumor cell lines (I.B. and A.R., unpublished observation). In contrast, the level of Mϕ induced inhibition of tumor cell proliferation correlates with the presence of substantial NO production in vitro (Fig. 4), as we previously reported (15). Blocking NO production inhibits killing, indicating that either NO itself is cytotoxic to tumor B cells or that the effects of NO on tumor or Mϕ (23, 24) populations are critical for tumor cell killing. There may exist a threshold level of NO necessary to allow for tumor cell killing, as Mϕ from older animals are fully capable of inducing tumor cell apoptosis despite secreting lower levels of NO upon activation as compared with Mϕ from young animals (Fig. 4). These findings are consistent with the reduced, though not absent, killing of solid tumor cell lines either by activated Mϕ from wildtype mice in the presence of l-NAME or by Mϕ from iNOS^-/- mice, as we recently reported (25). Interestingly, NO has been found to both augment and inhibit solid tumor growth and metastasis (26, 27). Macrophage-mediated killing may be another mechanism by which NO inhibits tumorigenesis, though there appears to be a complicated relationship between NO, iNOS, and inflammatory cytokines such as TNF-α and IFN-γ (28, 29), making it difficult to isolate the effects of NO.

In vivo, αCD40 plus CpG treatment is capable of inhibiting lymphoma cell growth in a T cell-independent fashion. αCD40 and CpG previously have been used separately in experimental tumor immunotherapy. Most of these approaches have focused on inducing T cell-mediated antitumor effects by giving either αCD40 (7) or CpG (30) to activate professional APC (or tumor cells in the case of B cell lymphoma). In our system, as in human B-CLL, CD40 cross-linking can augment the capacity of tumor B cells to activate T cells; however, this effect may be mitigated by intrinsic tumor-mediated suppression of T cell responses. The in vivo antitumor effect is evident in the immunocompromised SCID/Beige mice, which lack T, B, and cytotoxic NK cells, implicating Mϕ as the antitumor effector cells. Although other innate immune cells such as eosinophils and neutrophils may have cytotoxic activity, these do not express CD40 and thus would have to be activated indirectly by CD40-primed tumor cells or Mϕ in vivo. Neutrophils express TLR9 (31), however, and thus might be engaged in antitumor effects by treatment with CpG. We have not formally excluded this possibility in our Eμ-TCL1 tumor model, though given our in vitro results showing killing of even unstimulated tumor B cells by purified Mϕ, we favor Mϕ as the relevant effector cells in vivo. In addition, we previously demonstrated (14) that a combination of αCD40 and CpG resulted in marked inhibition of transplanted B16 tumor growth and prolonged survival in SCID/Beige mice that were additionally depleted of neutrophils and noncytotoxic NK cells that might secrete cytokines in response to αCD40 or CpG. Direct demonstration of the role of Mϕ in vivo in our model would be difficult, as repeated elimination of Mϕ by silica or clodronate-liposomes was found toxic when performed over the course of many weeks (I.B., unpublished observation). In addition, we would not be able to exclude that tumor B cells that could also phagocytize silica particles or clodronate-liposomes, would not be nonspecifically affected with this Mϕ-depleting approach.

During αCD40 plus CpG treatment, there is little or no Eμ-TCL1 B cell tumor growth. However, shortly after treatment cessation, the tumor cells grow again at the same apparent rate as those in the control treated group (Fig. 5A and data not shown). This is not surprising because activated Mϕ do not have any known mechanism for immunologic memory. In preliminary experiments, we found that the tumor cells growing in a SCID/Beige mouse after the cessation of αCD40 plus CpG treatment were equally responsive to αCD40 plus CpG treatment when transferred to naive SCID/Beige mice (data not shown), suggesting that tumor cells do not “escape” due to changes in susceptibility to activated Mϕ or to effects of CD40 and TLR9 signaling. In contrast, starting αCD40 plus CpG treatment in mice with high tumor burden has thus far been ineffective at slowing tumor progression (data not shown). This may be due either to the tumor cells acting as a sink for αCD40 and CpG, preventing adequate Mϕ stimulation, or simply to the overwhelming of the Mϕ system by excess tumor cells.

We previously demonstrated the in vivo effectiveness of αCD40 plus CpG treatment on CD40-negative solid tumors (14, 15). In contrast to these cell lines, the primary tumor cells in the current study express moderate levels of CD40 and can respond to the Ab (and CpG) directly, with increased proliferation and survival. For treatment of B cell (or other CD40-expressing) malignancies, the antitumor Mϕ-mediated effects will have to outweigh any progrowth properties of αCD40 and CpG. Our data demonstrate that αCD40 and CpG stimulate Eμ-TCL1 tumor B cells to proliferate and do not cause them to undergo apoptosis (Fig. 2), as expected. These results suggest that the in vivo antitumor effect of αCD40 plus CpG is unlikely to be due to direct killing of tumor cells by these agents. Ab-dependent cell cytotoxicity (ADCC) is one of the potentially important in vivo antitumor mechanisms of tumor-specific Abs. Other investigators have shown that despite the fact that Abs against numerous B cell surface Ags can mediate ADCC in vitro, only αCD40 appeared to be efficacious in vivo; and there was not a correlation between in vitro ADCC levels and in vivo effectiveness (21). Although we cannot exclude the possibility that ADCC may play a role in the antitumor effects of αCD40 in our system, it is clear from our current in vitro data (Fig. 3) wherein targeted B cells are not exposed to αCD40, that activated Mϕ do not require the lymphoma cells to bind Ab to eliminate them. It will be of interest in the future to determine whether other TLR ligands besides LPS (TLR 4) and CpG (TLR9) may provide better, or more specific activation of Mϕ, which express a wide range of other TLR, including TLR1, 2, 6-8, and 10 (32-36).

An intriguing question brought up by this study is how (and if) activated Mϕ might specifically direct their cytotoxic activity toward lymphoma cells in vivo. Unlike tumor cell lines growing rapidly in vivo, particularly in s.c. locations or in the lung, causing tissue damage and subsequent “danger” signals with concomitant local cytokine/chemokine production, these primary tumor B cells are slow growing and reside in the usual lymphoid/hematopoietic organs without creating a definable, tissue destructive “mass”. Although these tumor cells may fail to alert the innate immune system to their presence, they do have the ability to respond directly to both αCD40 and CpG. French et al. (7) found an increase in total body IL-12 and IFN-γ in αCD40 treated, BCL1-B cell lymphoma-bearing mice, but it was unclear whether tumor cells themselves were the source of these cytokines. CD40 and TLR9 stimulation may directly induce B cell IL-12 production (37) or indirectly increase IFN-γ and IL-12 production via a decrease in IL-10 production from the B cells (7, 38). In preliminary experiments (data not shown), we found that extended in vitro stimulation with LPS makes Eμ-TCL1 tumor B cells very potent activators of even naive Mϕ killing, though the mechanism has not been elucidated.

Several strategies based on manipulating tumor cells or APC through CD40 are now being developed for cancer treatment (39-41). These strategies, as initially conceived, are dependent on adequate T cell function. However, our findings support those of the Kipps’ laboratory in clinical trials of CD40L (CD154) transduced B-CLL cells in which peripheral leukemia counts and lymph node size rapidly dropped following infusion of CD154-expressing autologous tumor cells, suggesting an innate immune-based mechanism (39). Dicker et al. have suggested that this may be due to increased tumor B cell sensitivity to Fas-mediated apoptosis (41), but we could not show a role for Fas-mediated apoptosis in vitro in our model.

In most CLL patients (and in other cancers), T cell function is negatively impacted by tumor-derived cytokines (1, 2), chemotherapy (11), and advanced age. Our findings show that Mϕ can be effectively activated to mediate anti-lymphoma effects in the absence of other immune cells, suggesting that this immunotherapeutic strategy may be appropriate in immunocompromised cancer patients, particularly as Mϕ may recover more rapidly after adjuvant chemotherapy than other immune cells (42). Finally, Mϕ-mediated tumor cell killing may augment CD40-related strategies for developing potent T cell-based immunotherapy by providing an initial burst of apoptotic tumor cells for presentation by activated dendritic cells and Mϕ. This provides additional rationale for incorporating Mϕ activators such as CpG into αCD40-based immunotherapeutic strategies.