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Immunology |
1 Division of Surgical Oncology, University of Michigan Medical Center, Ann Arbor, Michigan; 2 Department of Surgery, Shiga University of Medical Science, Shiga, Japan; and 3 Bristol-Myers Squibb Company, Pharmaceutical Research Institute, Princeton, New Jersey
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ABSTRACT |
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 Top ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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) versus a type 2 (interleukin-4) profile. Cell depletion and the use of knockout animals identified that CD8+, CD4+, and NK cells were involved in the tumor rejection response and that CD8+ cells had the major effector role. Anti-CD137 administration resulted in increased proliferation of adoptively transferred OT-1 CD8+ T cells in the VPLNs of mice inoculated with B16-OVA TP-DCs. Polarization toward type 1 (interferon-) versus type 2 (interleukin-4) was also observed with the OT-1 cells from VPLNs and spleen cells after anti-CD137 injections. This polarization effect was abrogated by the in vivo depletion of NK cells. These findings indicate that the adjuvant effect of anti-CD137 given in conjunction with TP-DC vaccination is associated with the polarization of T effector cells toward a type 1 response to tumor antigen and is mediated via NK cells. |
INTRODUCTION |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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CD137 (4-1BB) is an important costimulating receptor that is expressed by activated natural killer (NK) cells, T cells, and DCs (11 , 12) . The natural ligand for CD137 is found on activated B cells, macrophages, and DCs. Ligation of CD137 results in cellular proliferation and protection of cells from activation-induced cell death (13 , 14) . CD137 ligation can be accomplished with agonistic monoclonal antibody (mAb) to CD137. Melero et al. (15) were the first to report that anti-CD137 mAb (anti-4-1BBmAb) can be administered in vivo to mediate antitumor responses against established weakly immunogenic tumors in animal models. Against poorly immunogenic or nonimmunogenic tumors, it was reported by the same group that tumor regression required active immunization with a peptide vaccine + anti-CD137 (16) . It was apparent from these and other studies that anti-CD137 administration can provide potent in vivo costimulation of cellular immune responses and may be a useful reagent to examine with DC-based vaccines.
In this report, we examined the role of anti-CD137 administration in modulating the immune responses induced by tumor lysate-pulsed DC (TP-DC) vaccinations. Tumor lysates provide the ability to sensitize T cells to the entire spectrum of tumor-associated antigens specific for an individual tumor. This has formed the rationale for clinical trials we and others have conducted (6 , 10) . The studies in this report involve the treatment of both weakly and poorly immunogenic established tumors. We describe herein the effects of anti-CD137 on the immune function of vaccine-primed T cells and how NK cells modulate this function.
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MATERIALS AND METHODS |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Tumors.
MCA 205 and MCA 207 murine tumors are 3-methylcholanthrene–induced fibrosarcomas, syngeneic to B6 mice. These tumors have been characterized previously as weakly immunogenic with distinct tumor-specific transplantation/rejection antigens (17)
. These tumors have been maintained in vivo by serial subcutaneous transplantation in B6 mice and were used within the 8th passage. Tumor cell suspensions were prepared from solid tumors by enzymatic digestion in 40 mL of Hanks’ balanced salt solution (Life Technologies, Inc., Grand Island, NY) containing 40 mg of collagenase, 4 mg of DNase I, and 100 units of hyaluronidase (all enzymes were from Sigma, St. Louis, MO) for 2 to 3 hours at room temperature, as described previously (18)
. Tumor cells were washed three times in Hanks’ balanced salt solution for administration to mice or resuspended in complete medium (CM) for in vitro assays. CM consisted of RPMI 1640 supplemented with 10% heat-inactivated fetal bovine serum, 0.1 mmol/L nonessential amino acids, 1 mmol/L sodium pyruvate, 2 mmol/L fresh L-glutamine, 100 mg/mL streptomycin, 100 units/mL penicillin, 50 µg/mL gentamicin, 0.5 µg/mL Fungizone (all from Life Technologies, Inc.), and 0.05 mmol/L 2-mercaptoethanol (Sigma).
The B16-BL6 melanoma is a poorly immunogenic melanoma of spontaneous origin (19) . B16-OVA is a tumor line transfected to express ovalbumin (OVA) that serves as a tumor-associated antigen. This line was obtained from Dr. Kenneth Rock (University of Massachusetts, Amherst, MA). EL-4 is a T-cell thymoma syngeneic to B6 mice. RMAS cells are TAP-deficient cells that are defective in transporters associated with antigen processing and express functionally "empty" class I major histocompatibility complex molecules on the cell surface (20) .
Antibody and Cytokines.
Agonistic anti-CD137 mAb (1D8) was kindly provided by Bristol-Myers Squibb Co., Pharmaceutical Research Institute (Princeton, NJ). ID8 is a rat IgG2a class of antibody. Recombinant murine granulocyte macrophage colony-stimulating factor and recombinant murine interleukin (IL)-4 were purchased from PeproTech (Rocky Hill, NJ).
Generation of Bone Marrow-derived Dendritic Cells and Tumor Lysate Pulsing.
Erythrocyte-depleted bone marrow cells were cultured in CM supplemented with 10 ng/mL granulocyte macrophage colony-stimulating factor and 10 ng/mL IL-4 at 1 x 106 cells per mL. On day 5, nonadherent cells were harvested by gentle pipetting. DCs were enriched by density centrifugation over 14.5% (w/v) metrizamide (Sigma), and the low-density interface was collected. DCs were washed twice, enumerated, and used for in vitro and in vivo functional studies. For preparation of tumor lysate, MCA 205, B16-BL6, B16-OVA, and EL-4 cells were suspended in PBS and subjected four times to rapid freeze-thaw exposures and then spun at 
100 x g for 5 minutes to remove cellular debris. The DCs were resuspended at 1 x 106 cells per mL in CM and incubated with tumor lysate at a tumor cell equivalent to DC ratio of 3:1 for 18 hours.
OT-1 Cell Purification and Labeling.
Freshly isolated splenocytes and popliteal, inguinal, mesenteric, axillary, and brachial lymph node (LN) cells from OT-1 transgenic mice (Ly5.1) were prepared as single cell suspensions and pooled. Lymphoid cells were enriched by the Magnetic Activated Cell Sorting device (Miltenyi Biotec, Auburn, CA), using the CD8a+ T-cell isolation kit according to the manufacturer’s protocol (Miltenyi Biotec). The fraction of tetramer-positive CD8+ T cells averaged between 93% and 97% of the total population by flow cytometry. Cells were suspended at 107 cells per mL in PBS and incubated with 5- (and 6)-carboxyfluorescein diacetate succinimidyl ester (CFSE; C-1157; Molecular Probes, Inc., Eugene, OR) at concentrations of 1 µmol/L for 10 minutes at room temperature in the dark and washed twice with PBS. Based on flow cytometry, a total lymphoid cell suspension containing 4 to 5 x 106 tetramer-positive CD8+ T cells was injected intravenously into C57BL/6 (Ly5.2) mice. To assess proliferation and CD62L expression of OT-1 cells in vaccine-primed lymph nodes (VPLNs), 0.5 to 1.0 x 106 events were collected and analyzed by flow cytometry.
Flow Cytometry.
The following antibodies were used in this study: purified rat anti-CD16/32 (2.4G2), fluorescein isothiocyanate (FITC)-labeled anti-CD3
(145-2C11), phycoerythrin (PE)-labeled anti-NK1.1 (PK136), FITC- and PE-labeled anti-CD4 (GK1.5: rat IgG2b), CyChrome anti-CD8a (53-6.7), PE-labeled anti-CD62L (Mel-14), PE- and biotin-labeled anti-CD45.1 (Ly5.1; A20), streptavidin CyChrome conjugate (all from BD PharMingen, San Diego, CA), and FITC-labeled anti-CD8a (5H10; rat IgG2b; Caltag, Burlingame, CA).
Before staining, cells were incubated with 2.4G2 mAb on ice for 15 minutes to prevent nonspecific binding of mAbs. All procedures were performed using cold PBS containing 5% fetal bovine serum. VPLN cell surface expression of CD137 was assessed by indirect immunofluorescence assays using purified anti-CD137 mAbs (1D8; rat IgG2a) followed by biotin-labeled antirat IgG2a (RG7/1.30), and purified rat IgG2a isotope standard (R35-95) was used as a control (both from BD PharMingen). To assess CD137 expression on NK cells and CD4+ and CD8+ T cells in VPLNs, 3 x 106, 0.5 x 106, and 0.5 x 106 events were collected in a FACScan flow cytometer, respectively. FACSCalibur with CellQuest software was obtained from Becton Dickinson (San Diego, CA).
Assessment of Cytokine Release in Response to Tumor Cells.
To measure cytokines released by VPLNs and spleen cells in response to tumor stimulation, 1.0 x 106 lymphoid cells were cocultured with 0.1 x 106 irradiated (6,000 rads) MCA 205 tumor cells or irradiated (10,000 rads) RMAS cells. RMAS cells were loaded with OVA–peptide257–264 (SINFEKL) at 10 µmol/L per mL in 2-mL volumes per well in 24-well tissue culture plates for 24 hours at 37°C. The culture supernatants were then collected and analyzed for cytokine production using ELISAs. Irradiated MCA 207 and RMAS cells without peptide were used as specificity controls.
Treatment of Established Pulmonary Metastases.
B6 mice received 2 x 105 MCA 205 or 1 x 105 B16-BL6 viable tumor cells intravenously on day 0. The mice were then immunized intradermally with 1.5 to 2.0 x 106 MCA 205, B16-BL6, or EL-4 TP-DCs on day 3 (treatment of 3-day–old lung metastases) or on days 10 and 14 (treatment of 10-day–old lung metastases). Anti-CD137 mAb (1D8) or control rat IgG (Sigma) was given intraperitoneally (100 µg) in 0.5 mL of PBS on days 4 and 7 (treatment of 3-day–old lung metastases) or on days 11, 14, and 17 (treatment of 10-day–old lung metastases). Mice were sacrificed on day 15 (for 3-day–old lung metastases) or 20 (for 10-day–old lung metastases) to enumerate lung metastases. Five mice were used in each experimental group. Intratracheal instillation with India ink was performed to stain the lung parenchyma (21)
. The MCA 205 metastases appeared as discrete white nodules on the black surface of lungs. For enumeration of B16-BL6 tumors, Fekete’s solution was instilled in the lungs via the trachea.
Treatment of Established Subcutaneous Tumor.
B6 mice were inoculated subcutaneously in the mid-right flank with 2 x 106 MCA 205 viable tumor cells on day 0. The mice were then immunized intradermally with 1.5 to 2.0 x 106 MCA 205 TP-DCs on days 7 and 11. Anti-CD137 mAb (1D8) or control rat IgG was given intraperitoneally (100 µg) in 0.5 mL of PBS on days 8, 11, and 14. The largest perpendicular diameters of tumors were measured in a blinded coded fashion using vernier calipers, and size was recorded as tumor area (in mm2). Data are reported as the average tumor area ± SE of six or more mice per group.
In vivo Cell Depletion.
Anti-NK1.1 hybridoma cells were used to produce the PK136 mouse IgG2a mAb (HB191; American Type Culture Collection, Manassas, VA). Hybridoma cells were inoculated into pristine-primed immunocompromised DBA/2 mice as described previously to generate ascites (22)
. MAb was purified from ascites fluid by ImmunoPure Immobilized Protein A/G gel (Pierce, Rockford, IL). The optimal amount of purified PK136 mAb for in vivo depletion was 400 µg intraperitoneally and was determined in preliminary functional assays of NK activity and flow cytometry (data not shown). The anti-CD4+ mAb (GK1.5; rat IgG2b) and the anti-CD8+ mAb (2.43; rat IgG2a) were purchased from Ligocyte (Bozeman, MT). The optimal amount of mAbs for in vivo depletion of CD4+ or CD8+ cells was determined in preliminary assays and found to be 800 and 400 µg intraperitoneally, respectively.
Statistical Analysis.
For comparison of treatment groups, a one-way analysis of variance (followed by a Newman-Keuls post hoc test) was performed using tumor measurements taken on the last day recorded for comparison of treatment groups. Student’s t test was used to analyze cell proliferation and cytokine release data. All statistical analysis was performed using GraphPad Prism software (San Diego, CA). Statistical significance was achieved when P was <0.05.
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RESULTS |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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, CD137 expression on CD3+ VPLN cells was negligible before vaccination. TP-DC vaccination resulted in 3.1% of CD3+ cells in the VPLNs expressing CD137 on day 3 compared with <1% of CD3+ cells in VPLN cells from animals inoculated subcutaneously with PBS or unpulsed DCs (UP-DCs). In the same experiment, CD3+ splenocytes from vaccinated mice did not have significantly altered CD137 expression at 3 or 7 days after vaccination (Fig. 1B)
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. The expression of CD137 in these subpopulations is depicted in Fig. 2B
. There was up-regulation of CD137 after TP-DC vaccination in all subpopulations compared with PBS-treated control mice. This was most notable in the CD3– NK1.1+ cells population. No CD137 up-regulation was observed on CD3+ NK1.1+ cells (data not shown).
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, mice treated with anti-CD137 alone or TP-DC vaccine had significantly reduced numbers of lung metastases compared with control animals. However, the combination of anti-CD137 + TP-DCs resulted in the greatest reduction of metastatic tumor, indicating an additive effect of the combined treatment. Using the nonimmunogenic B16-BL6 melanoma, we found that the combination therapy using anti-CD137 and TP-DCs was synergistic in mediating the regression of 3-day–old lung tumors (Fig. 3B)
. There was no antitumor effect with anti-CD137 alone or in combination with UP-DCs or EL-4 lysate-pulsed DCs. The latter represented a specificity control for B16-BL6. However, the combined therapy using anti-CD137 + B16-BL6 lysate-pulsed DCs resulted in significant tumor regression compared with all other groups.
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, immunotherapy using anti-CD137 alone or in combination with UP-DCs had no antitumor activity against 10-day–old metastases. Similarly, UP-DCs or TP-DCs alone showed no therapeutic activity. However, the combination of TP-DC vaccination + anti-CD137 administration resulted in significant tumor regression. This demonstrated a synergism between anti-CD137 and TP-DCs in generating an immune response within the host to reject advanced tumor burdens.
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, mice with 3-day–old lung metastases were treated with a single vaccination of TP-DCs and given either 50, 100, or 200 µg of anti-CD137. The lower dose of 50 µg was found to be less effective than the higher doses. In the 10-day–old lung model, the number of TP-DC vaccinations as well as multiple dosing of anti-CD137 (100 µg) was evaluated. As shown in Fig. 5B
, two TP-DC vaccinations + three doses of anti-CD137 resulted in better antitumor reactivity than one TP-DC vaccination + two doses of anti-CD137. Furthermore, increasing the number of vaccinations and doses of anti-CD137 did not improve the therapeutic effect.
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. However, the combined treatment of TP-DCs + anti-CD137 resulted in a significant reduction of tumor growth. This translated to significantly improved survival, as shown in Fig. 6B
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. Local tumor recurrence was the primary end point. By day 40, the group treated with TP-DCs + anti-CD137 had the lowest recurrence rate compared with the groups receiving monotherapy or control treatment (Fig. 7A)
. This group also had the best survival rate compared with the other groups (Fig. 7B)
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. VPLNs and spleens were harvested on day 9 for immune function analysis. Anti-CD137 administration alone resulted in no differences in interferon (IFN)- or IL-4 released by VPLNs or splenocytes compared with control animals. TP-DC administration alone resulted in the production of a modest amount of IFN- and IL-4 by both VPLNs and splenocytes in response to MCA 205 tumor cells. There was a significant increase in the amount of IFN- released by these cell populations from mice that received both TP-DCs and anti-CD137 (Fig. 8A)
. This response was immunologically specific. By contrast, the administration of anti-CD137 in conjunction with TP-DCs resulted in significantly decreased IL-4 release (Fig. 8B)
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Identification of the Effector Cells Mediating Tumor Regression In vivo.
We sought to identify the effector cells involved in the tumor rejection response associated with TP-DC + anti-CD137 therapy by using mAbs to deplete NK, CD4+, and CD8+ cells or by using KO mice. In antibody depletion experiments, the antitumor efficacy of TP-DC + anti-CD137 combined therapy was completely abrogated by the depletion of CD8+ T cells (Fig. 9A)
. Partial abrogation was observed by the depletion of CD4+ cells or NK cells. Use of KO mice devoid of NK, CD4+, or CD8+ cells confirmed this observation (Fig. 9B)
. The antitumor activity was completely abrogated in CD8 KO hosts and IL-15 KO hosts and partially abrogated in the CD4 KO mice. We concluded that the complete abrogation in the IL-15 KO mice was related to the absence of NK cells as well as an approximately 50% reduction of CD8+ cells in the IL-15–deficient mice compared with the wild-type host (data not shown). These data, taken together, indicate that CD8+ T cells are essential in the antitumor activity of TP-DC + anti-CD137, whereas NK cells and CD4 cells also play substantial roles.
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summarizes the cell yield obtained from VPLNs performed in four separate experiments. VPLNs were harvested 6 days after DC vaccination on day 0 followed by anti-CD137 administration on days 1 and 4. The administration of anti-CD137 alone did not result in an increase in the number of VPLN cells. The administration of UP-DCs or TP-DCs alone increased the cell yield of VPLNs compared non–DC-treated mice, but the values were not significantly different from each other. The combination of anti-CD137 and UP-DCs resulted in a VPLN cell yield that was not different from that seen with UP-DCs alone. However, the combination of anti-CD137 + TP-DCs resulted in a significantly higher cell yield in the VPLNs compared with TP-DCs alone or any other control groups. This suggested that the in vivo administration of anti-CD137 increased cell proliferation of antigen-reactive lymphoid cells that may have been sensitized by the TP-DC vaccine.
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Proliferation of the adoptively transferred OT-1 cells was assessed by measuring CFSE profiles of the VPLN cells using flow cytometry (Fig. 10)
. Mice given PBS, anti-CD137 alone, UP-DCs alone, or UP-DCs + anti-CD137 demonstrated minimal evidence of proliferation of OT-1 CD8+ T cells (10%). There was a minor increase in the number of CFSE-positive cells after TP-DC (14%). However, the combination of anti-CD137 + TP-DC resulted in significant proliferation of CFSE-labeled OT-1 cells in the VPLNs ( 34%). In addition, TP-DC + CD137 treatment increased the percentage of CD62Llow-expressing CFSE-labeled OT-1 cells (26%) compared with TP-DCs alone (11%), anti-CD137 alone (10%), and other control groups (
10%).
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, the administration of anti-CD137 + TP-DC significantly enhanced IFN- secretion by VPLNs and spleen cells to OVA compared with TP-DC + rat IgG. By contrast, antigen-stimulated IL-4 release by both cell populations was significantly reduced to undetectable levels when anti-CD137 was given in addition to TP-DC vaccination (Fig. 11B)
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, depletion of host CD4+ T cells significantly reduced IFN- secretion induced by anti-CD137 + DC but did not alter IL-4 production. However, after depletion of host NK1.1+ cells, the enhanced IFN- secretion and the decreased IL-4 secretion due to the use of anti-CD137 + DC were abrogated to levels comparable with those induced by DC vaccination alone. These results indicate that the antitumor efficacy of anti-CD137 + TP-DC combined therapy was associated with T-cell polarization toward a type 1 pattern mediated by NK cells, in addition to CD4+ T-cell help. |
DISCUSSION |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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We postulated that anti-CD137 administration may potentiate the antitumor reactivity of TP-DC therapy. Subsequent experiments demonstrated that administration of anti-CD137 enhanced the antitumor reactivity of TP-DC vaccines in both weakly (i.e., MCA 205) and poorly (i.e., B16-BL6) immunogenic tumor models. The combined therapy of anti-CD137 + TP-DC resulted in more effective therapy of advanced disease and improved survival and was effective in the postoperative setting in treating residual microscopic disease.
We found that anti-CD137 administration polarized the antitumor reactivity of T cells to tumor antigen toward a type 1 response (i.e., IFN-) and away from a type 2 response (i.e., IL-4). This was observed in the VPLNs and spleens of vaccinated mice. The results of this study extend our previous observations regarding the ability of anti-CD137 to polarize T-cell reactivity during in vitro culture (26)
. In that report, we examined the effects of anti-CD3, anti-CD28, and anti-CD137 on activating tumor-draining lymph node (TDLN) cells in vitro. Anti-CD3, with or without anti-CD28, up-regulated CD137 expression on TDLN cells. The subsequent addition of anti-CD137 polarized the type 1 versus type 2 response of the TDLN cells and augmented their therapeutic efficacy in the adoptive immunotherapy of established tumor.
The in vivo antitumor reactivity of anti-CD137 + TP-DC vaccination was mediated by CD8+, CD4+, and NK cells. Among these lymphoid cells, CD8+ T cells manifested the most significant antitumor effect as compared with CD4+ and NK cells. Other reports examining anti-CD137 alone as an antitumor therapy have also found CD8+ cells to be critical (15 , 27) . The major role of CD8+ cells as effectors may relate to the relatively greater expression of CD137 on CD8+ cells compared with CD4+ cells after T-cell receptor engagement (28) . The depletion of CD4+ or NK cells partially abrogated the antitumor response of therapy, which suggested that these populations may have an interactive role with CD8+ T cells. In adoptive immunotherapy models, CD4+ tumor reactive cells have been shown to provide help to CD8+ cells by releasing IL-2 (22) . Wilcox et al. (29) recently reported that CD137-stimulated NK cells promote the expansion of CD8+ cytolytic T cells in vitro, thus providing another form of help.
We consistently found that TP-DC vaccines + anti-CD137 resulted in a greater cell yield from the VPLNs compared with UP-DC vaccine given with or without anti-CD137. To further investigate the mechanisms of this effect, we used the OT-1 transgenic model. We found that the increased number of cells in the LNs draining TP-DCs was due to the preferential accumulation of antigen-reactive OT-1 cells. These cells also demonstrated increased proliferation compared with OT-1 cells found in LNs draining UP-DCs. In addition, we found that the administration of anti-CD137 caused increased survival and proliferation of antigen-reactive cells (i.e., OT-1 CD8+ cells). Several groups have reported previously that anti-CD137 can promote survival of CD8+ T cells (30, 31, 32, 33, 34) . The induced proliferation of CD8+ cells by anti-CD137 is supported by some investigators (30, 31, 32, 33) but not by others (34) . The majority of studies and our data would support the former.
The polarization effect of anti-CD137 was also observed in the OT-1 model. The depletion of NK cells, but not CD4+ cells, reversed the polarization effect of the cytokine response of both VPLNs and splenocytes. These findings demonstrate a novel interaction between anti-CD137–activated NK cells and T cells. The cross-talk between NK cells and CD8+ cells that promotes expansion and increased lytic activity as reported by Wilcox et al. (29) may be related to the cytokine milieu seen in our studies; namely, the down-regulation of type 2 cytokines.
In summary, there are several salutary effects of anti-CD137 when administered in conjunction with tumor vaccines. These include polarization of tumor-reactive T-cell responses toward a type 1 profile, increased survival and proliferation of activated T cells, and activation of NK cells that interact with the adaptive immune system in the polarization process. This provides a useful tool to break immunologic "ignorance" to poorly immunogenic tumors, making the combined therapy using this antibody and TP-DC vaccine more effective.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
Requests for reprints: Alfred E. Chang, 3302 Cancer Center, 1500 East Medical Center Drive, Ann Arbor, MI 48109. E-mail: aechang{at}umich.edu
Received 2/18/04. Revised 9/ 1/04. Accepted 9/ 8/04.
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REFERENCES |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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