Abstract
Allogeneic bone marrow transplantation (BMT) causes a beneficial graft-versus-tumor (GVT) immune response that is often associated with graft-versus-host disease (GVHD). There is substantial interest in developing therapeutic strategies that augment GVT without GVHD. We have demonstrated recently that immunization of BMT donors with cellular tumor vaccines leads to curative GVT but induces unacceptable GVHD because of the presence of recipient minor histocompatibility antigens (mHAgs) in whole-cell tumor vaccines. This study tested the hypothesis that immunization of BMT donors against a defined tumor-specific antigen with a vaccine not containing recipient mHAgs would help to separate the two responses by enhancing GVT activity without exacerbating GVHD, even when cellular vaccines were used after BMT. Recipient strain C57BL/6 fibrosarcoma cells engineered to express the well-characterized model tumor antigen,influenza nucleoprotein (NP), were used in these studies. C3H.SW donors were immunized against NP prior to BMT, and cytolytic T cells were transferred along with bone marrow into irradiated H-2-matched,mHAg-mismatched C57BL/6 recipients with established micrometastatic 205-NP tumors. Donor immunization led to a significant increase in GVT activity, as measured by reduction in tumor growth and enhanced survival. However, deaths in recipients of tumor antigen-specific immune BMT ultimately occurred because of the growth of antigen-loss variants; such tumor growth did not occur in animals receiving BMT from donors treated with whole-cell vaccines. Donor immunization did not lead to an exacerbation of GVHD, even when BMT recipients received additional immunization after BMT with a 205-NP “whole” tumor cell vaccine (which was shown to induce fatal GVHD when used for donor immunization). In conclusion, immunization of allogeneic BMT donors against a tumor-specific antigen significantly enhances GVT activity without an associated exacerbation of GVHD.
INTRODUCTION
Tumor vaccine strategies require host immunocompetence for effectiveness. However, cancer patients often have impaired immune competence because of protracted chemotherapy and/or tumorinduced immune suppression (1, 2, 3, 4, 5). In allogeneic BMT,3donor T lymphocytes have not been tolerized by tumor cell products or chemotherapy and are transferred to patients with minimal residual tumor burden. This setting may be more conducive to successful cancer immunotherapy. Indeed, allogeneic BMT is associated with a GVT immune response, but this response may be even more powerful if a tumor vaccine is used to prime donor lymphocytes against tumor antigens prior to BMT (6, 7, 8).
We have shown previously that allogeneic BMT donor immunization using recipient-derived “whole” tumor cell vaccines produces tumor-curative GVT activity but also produces unacceptable GVHD(9). We have also demonstrated that this increase in GVHD is attributable to the presence of immunodominant mHAgs on the“whole” tumor cell vaccines (9, 10). However, some donor T cells mediating GVT activity have been shown to be tumor specific and distinct from those mediating GVHD (6, 9, 11, 12). In theory, if donors could be immunized against a tumor-specific antigen without simultaneously being immunized against mHAgs, it is conceivable that one could potentiate the GVT activity without exacerbating GVHD.
Recently, there has been substantial progress in the molecular identification of human tumor antigens (13, 14, 15). These developments have created opportunities for selective immunization using recombinant proteins, peptides, or even nucleic acid vaccines without the use of “whole” cell vaccines, which induce responses to mHAgs as well as tumor antigens (16, 17).
Less is known about murine tumor antigens in well-characterized allogeneic BMT systems. Therefore, to explore the biological effects of immunizing allogeneic donors against molecularly defined tumor antigens, we used an established model tumor antigen system, the NP from the influenza A virus. The NP gene has been cloned, and murine tumor cell lines have been modified to express this gene (18, 19). Tumor cells expressing the NPgene grow progressively without loss of antigen in immunocompetent syngeneic mice (19). Furthermore, the immunodominant MHC class I-associated NP peptide recognized by CD8+T cells has been identified and can be used to study NP-specific cytolytic T cell responses in vitro (20, 21).
It is possible that the relative potency of GVT activity could be increased by using donor immunization to activate and expand donor T cells capable of recognizing tumor antigens prior to BMT. An additional possibility is that donor immunization to the defined antigen would adversely affect GVHD because activated donor T cells secrete proinflammatory cytokines (e.g., IFN-γ), which may play a role in the pathogenesis of GVHD. The experiments described in this study tested the hypothesis that immunization of immunocompetent MHC-matched donors against a model tumor-specific antigen would increase GVT activity and extend survival of BMT recipients bearing preexisting micrometastatic tumor without exacerbating GVHD.
MATERIALS AND METHODS
Animals
Female C57BL/6 (B6) mice were purchased from the National Cancer Institute (Frederick, MD), and female C3H.SW-H2b/SnJ (C3H.SW or SW)mice were purchased from The Jackson Laboratory (Bar Harbor, ME). They were used for experiments at 6–12 weeks of age. Mice were housed in conventional rooms with food and water ad libitum and were cared for by the Department of Veterinary Medicine and Surgery at M. D. Anderson Cancer Center. From 2 or 3 days prior to BMT until day 14, water was acidified (pH 2.5) and supplemented with 2 g/l neomycin sulfate (Sigma Chemical Co., St. Louis, MO).
Cell Lines
205 is a weakly immunogenic, methylcholanthrene-induced C57BL/6 fibrosarcoma cell line (22). This tumor is not spontaneously metastatic but reproducibly forms multiple lung nodules when at least 1 × 104 cells are injected i.v. into C57BL/6 mice. 205-NP is a 205 cell line modified to express the NP gene from the influenza A/PR/8/34 virus using the LXSN retroviral vector (23). 205-B7-NP is a 205 cell line transduced with the B7.1 immune costimulatory molecule (LXSN vector) and also transfected with the NP gene (BP-NP-I-H vector). The LXSN vector contains a neomycin resistance gene, and the BP-NP-I-H vector carries a hygromycin resistance gene. Transduced cells were selected in 300 μg/ml hygromycin and/or 1 mg/ml G418. EL4 is a C57BL/6 lymphoma cell line (American Type Culture Collection,Rockville, MD), and B16 is a spontaneous, weakly immunogenic C57BL/6 melanoma cell line (a gift from Dr. I. J. Fidler, M. D. Anderson Cancer Center). Cells were grown in tissue culture using RPMI 1640 supplemented with 5% heat inactivated fetal bovine serum(BioWhittaker, Walkersville, MD) and 2 mml-glutamine.
BMT Donor Immunization against NP Antigen
Influenza Vaccine.
Human influenza A virus (strain A/PR/8/34) was obtained from American Type Culture Collection (Rockville, MD) and expanded by passage through embryonated chicken eggs. C3H.SW BMT donors (Ag-Immune SW) were immunized by i.p. injection of 5 × 106 influenza virus-infected C3H.SW splenocytes. Splenocytes were infected by incubation for 1 h at 37°C with live influenza virus on a rocker at a cell concentration of 20 × 106 cells/ml in RPMI 1640. Donor immunization was repeated 2 weeks after the first injection.
Irradiated Tumor Cell Vaccine.
C3H.SW donors (Tumor-Immune SW) were injected s.c. in the flank with 5 × 106 50 Gy-irradiated C57BL/6 205-NP tumor cells in 0.2 ml of HBSS. The immunization was repeated 2 weeks after the first injection.
BMT
BMT recipients (C57BL/6) received 850 cGy TBI using a 60Co source 1 day before BMT. On the day of BMT,2–4 × 106 bone marrow cells and 5–10 × 106 spleen cells were injected i.v. together in a total volume of 0.2 ml of HBSS. Bone marrow was isolated from donors (C3H.SW) by flushing each femur and tibia with RPMI 1640. Spleen cells were isolated by macerating spleens between two frosted glass slides, followed by lysis of erythrocytes. Mice that died from sepsis during the first 10 days after BMT (<10%) were excluded from these studies.
BMT Recipient NP Antigen Immunization
Some recipients were treated once by injection of 5 × 106 irradiated 205-NP cells on the day of BMT. In other experiments, some recipients were injected with 5 × 106 irradiated 205-B7-NP cells s.c. in 0.2 ml HBSS four times at weekly intervals starting the day after BMT.
Pulmonary Tumor Assays
Micrometastatic lung tumors were established by injecting C57BL/6 mice with 1 × 105 205-NP tumor cells i.v. in 0.2 ml of HBSS 6 days prior to BMT. After death or sacrifice, lungs were stained black by suffusion with India ink instilled through the trachea. Lungs were fixed, and white tumor nodules on the black lung surface were counted without magnification.
Analysis of Antigen Expression in Pulmonary Tumors
After sacrifice, the thoracic cavity was opened in a sterile manner to visualize the surface of the lungs. Individual lung nodules were excised from the lung surface, macerated between two frosted glass slides, and placed in tissue culture with complete medium. They were stained for NP expression using the PT107 monoclonal antibody (mouse IgG2b anti-NP; a kind gift of Dr. J. Schulman, Mount Sinai Medical Center, New York, NY) after fixing and permeabilizing the cells with a 1:1 mixture of cold acetone and methanol. A FITC-labeled goat antimouse secondary antibody was used for detection. Controls included unmodified 205 tumor cells labeled with both antibodies and cells incubated with the secondary antibody only. Flow cytometric analysis was performed using a FACScan and Lysis II software (Becton Dickinson, Moutainview,CA).
Evaluation of GVHD
Recipients were weighed weekly and observed daily for signs of GVHD (weight loss, alopecia, dermatitis, hunched posture, and death). In some experiments, histological examination of livers for GVHD was performed. Liver sections stained with H&E were examined for mononuclear infiltrates in portal triads characteristic of GVHD.
Cytotoxicity Assays
For use as effector cells, spleen cells were cultured in six-well plates at 1 × 106cells/ml and 10 ml/well in RPMI 1640 supplemented with 10% FBS (Summit Biotech, Ft. Collins, CO), 100 units/ml penicillin, 100 μg/ml streptomycin, 2 mm l-glutamine, 100 mm sodium pyruvate, 0.1 mm nonessential amino acids, and 50 μm 2-mercaptoethanol (complete medium). Stimulator cells were loaded with NP366 peptide (ASNENMETM) by incubation for 1.5 h at 37°C with 35 μg/ml peptide(synthesized by the Peptide Synthesis Core Lab of M. D. Anderson Cancer Center). After 5 days in culture with stimulator cells, effector cells were harvested and plated in triplicate with 5 × 103 51Cr-labeled target cells/well at E:T ratios ranging from 200:1 to 12.5:1. 205-NP target cells were labeled by combining 5 × 106 cells in 0.1 ml of complete medium with 0.1 ml (∼100 μCi) sterile isotonic Na251CrO (Amersham, Arlington Heights, IL) for 60 min at 37°C. Labeled targets were washed three times before plating with effectors in a total volume of 0.2 ml/well in 96-well, round-bottomed plates. Plated cells were incubated for 4 h at 37°C, after which 0.1 ml of supernatant was counted in a gamma counter (Wallac, San Francisco, CA). The percentage of lysis was calculated as: 100 × [(experimental cpm − spontaneous cpm)/(maximum cpm − spontaneous cpm)]. Spontaneous release was usually <20% and always less than 30% of the maximum release.
Statistical Analysis
Prism 3.0 software (GraphPad Software for Scientists, Sorrento,CA) was used for statistical evaluation of data. When more than two groups were compared, a one-way ANOVA was performed. If P < 0.05 overall, then the groups were compared using a Tukey’s multiple comparison test. When only two groups were compared, a Student’s t test was used. To compare Kaplan-Meier survival curves, the log-rank test was used. For regression analysis of NP+ cells, Statistica for Windows 5.1 (StatSoft,Tulsa, OK) was used using the percentage of flow cytometry-positive cells and the percentage of cytolysis as continuous variables.
RESULTS
Tumor Antigen-specific Immunity from BMT Donors Can Be Transferred to Allogeneic BMT Recipients Treated with a Cellular Tumor Vaccine at the Time of BMT.
Experiments were first performed to determine whether a cytolytic T cell-mediated immune response to the model tumor antigen could be efficiently transferred to allogeneic BMT recipients. Donors (Ag-Immune C3H.SW) were selectively immunized against influenza virus to induce immunity to the NP model tumor antigen, and both bone marrow and spleen cells were transplanted into irradiated allogeneic C57BL/6 recipients. Other experiments in a syngeneic BMT model (C57BL/6) had shown that peritransplant recipient exposure to NP antigen was required for efficient transfer of the cytolytic immune response (data not shown). Some C57BL/6 BMT recipients were exposed to NP antigen on the day of BMT by injection of 5 × 106irradiated whole 205-NP tumor cells. Three weeks after BMT, CTL activity against the immunodominant MHC class I-presented NP peptide was assessed by in vitro NP peptide restimulation and chromium release assay using NP+ tumor cells as targets. As seen in Fig. 1, potent CTL activity was present in recipients of immune donor cells that had been exposed to antigen at the time of transplant. The cytolytic activity was NP specific. C57BL/6 EL4 lymphoma cells were not lysed unless they were preincubated with NP peptide (Fig. 1 B). The cytolytic cells also failed to lyse B16, another C57BL/6 tumor that does not express NP (data not shown). The cytolytic activity in recipients of Ag-immune cells exposed to NP at the time of BMT was not a primary immune response. In other experiments, we have observed that recipients of cells from naive donors are incapable of mounting a primary anti-NP cytolytic response until 5 weeks after BMT(data not shown).
Transfer of Tumor Antigen-specific Immunity from Allogeneic BMT Donors Decreases Tumor Burden in Recipients with Preexisting Micrometastatic Cancer.
We have shown recently that immunization of donors with whole tumor cell vaccines induces curative GVT activity against preexisting micrometastatic disease (9, 10). Although the experiments described above indicate that tumor antigen-specific CTL cells could be efficiently transplanted, this result did not prove that such antigen-specific immunity without additional reactivity directed against mHAgs could prove effective in vivo against preexisting metastatic disease. To test this possibility, donors(C3H.SW) were selectively immunized against the NP tumor antigen(Ag-Immune) by exposure to syngeneic C3H.SW spleen cells infected with influenza virus. Control donors were immunized with irradiated 205-NP tumor cells (Tumor-Immune) that induce immunity to both tumor antigen and allogeneic mHAgs. Donor bone marrow and spleen cells were transplanted into irradiated C57BL/6 recipients with preexisting micrometastatic 205-NP pulmonary tumors. Recipients of NP-immune(Ag-Immune) donor BMT exhibited both prolonged survival(P = 0.018) and a significant reduction in pulmonary tumor nodules (P < 0.01) compared with recipients of Non-Immune donor BMT (Fig. 2). None of these BMT recipients had GVHD. However, all recipients of Ag-Immune BMT died from progressively growing lung tumors. This result was in contrast to Tumor-Immune donor BMT, which completely eradicated growth of pulmonary tumor nodules in most recipients but also induced fatal GVHD in nearly all recipients.
Lung Metastases Growing in Recipients of Tumor Antigen-immune Donor BMT Are Antigen-Loss Variants.
Because most but not all lung nodules were prevented from growing in recipients of Ag-immune BMT, we hypothesized that the reason for progressive tumor growth was loss or down-regulation of NP antigen expression. Although Tumor-Immune donor lymphocytes from donors treated with whole tumor cell vaccines can recognize both the NP antigen and C57BL/6 minor histocompatibility antigens on 205-NP cells (9, 10), the only antigen on 205-NP tumor cells recognized by influenza virus-immune donor lymphocytes is the NP antigen. Therefore,loss of NP expression could cause evasion from the immune attack. To test this hypothesis, progressively growing lung nodules from recipients of Ag-Immune BMT and control nodules from recipients of NonImmune donor BMT were randomly selected and removed 1 month after BMT. As seen in Fig. 3, all tumors reisolated from Ag-Immune BMT recipients had complete or nearly complete loss of expression of the NP antigen as assessed by flow cytometry. In contrast, most of the lung nodules reisolated from Non-Immune BMT recipients, where there was no transplanted selective pressure for antigen loss, expressed NP at substantially higher levels.(Several tumor reisolates from nonimmune recipients had reduced levels of NP expression; this may have resulted from primary anti-NP immune responses in the recovering immune system.) The observed reduction in NP expression was not the result of short-term growth in vitro in the absence of neomycin selection. 205-NP was grown in continuous culture for 28 days in the absence of G418; at the end of this time, 81% of the cells were positive by flow cytometry for NP expression compared with 91% at the beginning of the month in culture(data not shown). Cells that do not express NP as measured by flow cytometry are not sensitive to NP-specific CTLs. In an independent experiment, 205-NP tumors were reisolated from naive or influenza immune C57BL/6 mice and analyzed for NP expression and sensitivity to NP-specific CTLs. The positive control 205-NP tumor was 78% positive for NP by flow, and the percentage of lysis was 36% at an E:T ratio of 130; the negative control 205 had undetectable antigen (2%) and 8%lysis. NP expression in tumors from influenza immune hosts(n = 2) had undetectable NP expression (1%),and lysis ranged from 0–5%. In contrast, in 205-NP resiolates from nonimmune hosts (n = 5), NP expression ranged from 20 to 53%, and lysis ranged from 7 to 39%. Regression analysis demonstrated a statistically significant relationship(P < 0.016) between NP expression by flow cytometry and sensitivity to anti-NP CTLs.
Transfer of Tumor Antigen-specific Immunity from Allogeneic BMT Donors Does Not Exacerbate GVHD.
Recipients of Ag-Immune allogeneic donor BMT in these experiments did not exhibit signs of GVHD. Instead, they died from progressive tumor growth by day 40. However, because in this BMT model GVHD may not become evident until after that time, early tumor growth may have prevented detection of GVHD. Active immune responses after BMT(e.g., to viral infections) have been associated with exacerbations of GVHD (24); therefore, it remained a possibility that transfer of an active T-cell response to non-alloantigen could exacerbate GVHD. Additional experiments were conducted to determine the effect of tumor-specific immunity on the development of GVHD in recipients without the confounding factor of progressively growing tumor. C3H.SW donors were immunized with either influenza virus (Ag-Immune SW) or irradiated 205-NP tumor cells(Tumor-Immune SW). Control donors were not immunized (Non-Immune SW). Donor bone marrow and spleen cells were transplanted into lethally irradiated C57BL/6 mice without tumors. Although Tumor-Immune(allo-immune) control donor lymphocytes induced fatal GVHD, Ag-Immune donor cells did not induce signs of GVHD in any of the recipients(n = 10; Fig. 4,A). The capacity of NP Ag-Immune donor cells to cause GVHD was further tested in a second experiment in which some recipients were actively immunized with irradiated 205-B7-NP tumor cells that express both NP and C57BL/6 mHAgs. (Tumor cells coexpressing NP, mHAgs, and the B7.1 costimulatory molecule were chosen to help avoid the issue of a deficiency of antigen-presenting cells in the peritransplant period.)In this setting, it is conceivable that the anti-NP response could induce cytokines that cross-prime a response to the mHAgs and worsen GVHD. However, even when recipients were exposed to NP antigen on the same cells with mHAgs after BMT, there was no increase in mortality(Fig. 4 B) or other measures of GVHD (death, weight loss, fur loss, or dermatitis) compared with recipients of nonimmune donor BMT.
DISCUSSION
This work demonstrated that immunization of MHC-matched allogeneic donors against a tumor-specific antigen with vaccines that do not contain recipient mHAgs can enhance GVT activity and prolong survival of BMT recipients with preexisting micrometastatic disease without exacerbation of GVHD. This was also the case in experiments in which BMT recipients received additional post-BMT vaccination with whole tumor cell vaccines that contain recipient mHAgs. This is in contrast to our observations published recently that immunization of BMT donors with whole tumor cell vaccines, although highly effective in enhancing GVT activity, leads to severe GVHD (9). Our results are compatible with reports by others in which use of tumor antigen-specific vaccines in conjunction with autologous BMT leads to enhanced antitumor immune activity (25).
Although donor immunization produced a significant increase in GVT activity that extended survival and reduced the number of pulmonary tumor nodules growing in recipients (Fig. 2), the GVT activity was not curative. Flow cytometry demonstrated that the lung metastases that emerged in antigen-immune recipients had either down-regulated or lost their expression of the NP gene (Fig. 3). Although BMT from nonimmune donors did not eliminate NP expression, NP-immune donor cells most likely caused a selective growth advantage for antigen-loss variants. NP antigen expression in the tumor challenge population was heterogeneous (Fig. 3 A), reflecting the antigenic heterogeneity of clinical tumors. Antigen-loss variants recovered in vivo could be outgrowths of low- or nonexpressing cells injected, or they could be antigen-loss variants generated in vivo (26, 27). The observation of antigen-loss variants in this model indicates that effective vaccines will need to be directed against more than one tumor-specific antigen.
Recipients of antigen-immune BMT did not develop GVHD, even when exposed to NP antigen and allogeneic mHAgs multiple times by weekly s.c. injection of irradiated 205-B7-NP tumor cell vaccines. In no experiment was there an increase in the incidence or mortality of GVHD compared with recipients of nonimmune donor cells (Fig. 4). Although GVHD can be exacerbated in an antigen-nonspecific manner by the cytokine release induced by BMT preparative regimens (28)or viral infections (24), these experiments suggest that the proinflammatory activity of the induced T cells did not contribute in an antigen-nonspecific manner to GHVD.
Despite the problem of antigen-loss variant tumor growth using the tumor antigen-specific vaccination strategy, these results suggest potentially useful strategies for BMT patients. One approach would be the simultaneous immunization of donors with several tumor antigens or antigens with multiple epitopes to make variant selection in vivo more difficult. Transfer of a polyclonal T-cell response to tetanus toxoid from allogeneic donors boosted with tetanus vaccines prior to cell harvest has been demonstrated in clinical transplantation(29). For cancer, this approach will require identification of multiple antigens in the same tumor. Although tumor-specific antigens for many malignancies have not yet been identified, several have been molecularly characterized for melanoma and other cancers (16, 17, 30, 31, 32). In addition, tumor specific idiotypes from myelomas can be molecularly characterized and treated as tumor antigens; the clinical feasibility of using such idiotypes as vaccines in conjunction with autologous transplantation has been established (33, 34, 35). An alternate and possibly more feasible approach is immunization of donors with one defined tumor antigen, followed by post-BMT vaccination of recipients with a“whole” cell tumor vaccine that may contain other target antigens that have not been molecularly identified. Our recent work(10) and data here show that post-BMT immunization of recipients with “whole” tumor cell vaccines does not exacerbate GVHD; therefore, such a combined strategy would potentially allow multiple tumor antigens to stimulate a more powerful GVT immune response with reduced probability of provoking GVHD against mHAgs. Our current work explores such strategies.
Anti-NP immunity can be efficiently transferred to allogeneic BMT recipients treated with tumor vaccine at the time of BMT. C3H. SW (SW) donors were either immunized against NP with influenza virus-infected syngeneic spleen cells(Ag-Immune SW) or not immunized (Non-Immune SW). Two weeks later, their bone marrow cells (4 × 106) and spleen cells (10 × 106) were transplanted into C57BL/6 mice treated with 850 cGy TBI 1 day before BMT. Some Ag-Immune SW and Non-Immune SW were not used for BMT and were not further manipulated until they were used as controls for the cytotoxicity assay 3 weeks after BMT. One group of recipients was exposed to NP antigen by s.c. injection of 5 × 106 irradiated 205-NP tumor cells on the day of transplant (+ NP Ag Day 0), whereas the other group was not exposed to NP antigen (No NP Ag). Three weeks after BMT, splenocytes were isolated and stimulated for 5 days in vitro with NP peptide-loaded C3H. SW splenocytes before testing them for cytolytic activity against NP+ cells. In experiment A, 205-NP tumor cells were used as the NP+ target. Syngeneic B16 melanoma cells, which do not express NP, were the negative control, and lysis of these targets was <10% (data not shown). In experiment B, C57BL/6 EL4 cells pulsed with MHC-binding NP peptide (NP-pulsed EL4) were the NP+target, whereas EL4 cells not incubated with NP peptide were the negative control. Each E:T condition was performed in triplicate using splenocytes pooled from two or three mice/group. Bars,SE.
Anti-NP immunity can be efficiently transferred to allogeneic BMT recipients treated with tumor vaccine at the time of BMT. C3H. SW (SW) donors were either immunized against NP with influenza virus-infected syngeneic spleen cells(Ag-Immune SW) or not immunized (Non-Immune SW). Two weeks later, their bone marrow cells (4 × 106) and spleen cells (10 × 106) were transplanted into C57BL/6 mice treated with 850 cGy TBI 1 day before BMT. Some Ag-Immune SW and Non-Immune SW were not used for BMT and were not further manipulated until they were used as controls for the cytotoxicity assay 3 weeks after BMT. One group of recipients was exposed to NP antigen by s.c. injection of 5 × 106 irradiated 205-NP tumor cells on the day of transplant (+ NP Ag Day 0), whereas the other group was not exposed to NP antigen (No NP Ag). Three weeks after BMT, splenocytes were isolated and stimulated for 5 days in vitro with NP peptide-loaded C3H. SW splenocytes before testing them for cytolytic activity against NP+ cells. In experiment A, 205-NP tumor cells were used as the NP+ target. Syngeneic B16 melanoma cells, which do not express NP, were the negative control, and lysis of these targets was <10% (data not shown). In experiment B, C57BL/6 EL4 cells pulsed with MHC-binding NP peptide (NP-pulsed EL4) were the NP+target, whereas EL4 cells not incubated with NP peptide were the negative control. Each E:T condition was performed in triplicate using splenocytes pooled from two or three mice/group. Bars,SE.
Transfer of tumor antigen-specific immunity from allogeneic BMT donors decreases tumor burden in recipients with preexisting micrometastatic cancer. C3H.SW (SW) donors were immunized with 5 × 106 influenza virus-infected syngeneic spleen cells i.p. (Ag-Immune SW) or 5 × 106 irradiated 205-NP tumor cells s.c. (Tumor-Immune SW), and immunizations were repeated 2 weeks later. Control donors were not immunized(Non-Immune SW). Two weeks after the second vaccine,donor bone marrow cells (4 × 106) and spleen cells (10 × 106) were transplanted into C57BL/6 mice with 205-NP lung micrometastases established by i.v. injection of 1 × 105 205-NP cells 6 days prior to BMT. Recipients underwent 850 cGy TBI 1 day before BMT.∗∗, recipients of either Ag-Immune or Tumor-Immune donor BMT exhibited increased survival (P = 0.0180)compared with recipients of Non-Immune donor BMT. ∗∗∗, recipients of Ag-Immune or Tumor-Immune donor BMT had a significant reduction in pulmonary tumor nodules (P < 0.01)compared with recipients of Non-Immune donor BMT. Recipients of Ag-Immune BMT died from progressively growing lung tumors. Recipients of Tumor-Immune BMT often had complete prevention of pulmonary tumor nodule growth but developed fatal GVHD. This experiment is representative of three independent experiments in which Ag-Immune BMT caused significant but incomplete protection against 205-NP lung nodule growth. In this experiment, each group had a sample size of 4. Bars, SE.
Transfer of tumor antigen-specific immunity from allogeneic BMT donors decreases tumor burden in recipients with preexisting micrometastatic cancer. C3H.SW (SW) donors were immunized with 5 × 106 influenza virus-infected syngeneic spleen cells i.p. (Ag-Immune SW) or 5 × 106 irradiated 205-NP tumor cells s.c. (Tumor-Immune SW), and immunizations were repeated 2 weeks later. Control donors were not immunized(Non-Immune SW). Two weeks after the second vaccine,donor bone marrow cells (4 × 106) and spleen cells (10 × 106) were transplanted into C57BL/6 mice with 205-NP lung micrometastases established by i.v. injection of 1 × 105 205-NP cells 6 days prior to BMT. Recipients underwent 850 cGy TBI 1 day before BMT.∗∗, recipients of either Ag-Immune or Tumor-Immune donor BMT exhibited increased survival (P = 0.0180)compared with recipients of Non-Immune donor BMT. ∗∗∗, recipients of Ag-Immune or Tumor-Immune donor BMT had a significant reduction in pulmonary tumor nodules (P < 0.01)compared with recipients of Non-Immune donor BMT. Recipients of Ag-Immune BMT died from progressively growing lung tumors. Recipients of Tumor-Immune BMT often had complete prevention of pulmonary tumor nodule growth but developed fatal GVHD. This experiment is representative of three independent experiments in which Ag-Immune BMT caused significant but incomplete protection against 205-NP lung nodule growth. In this experiment, each group had a sample size of 4. Bars, SE.
205-NP Lung nodules growing in recipients of Ag-Immune donor BMT exhibit loss of NP antigen expression. C3H.SW(SW) donors were immunized with 5 × 106 influenza virus-infected syngeneic spleen cells i.p.(Ag-Immune SW) or were not immunized (Non-Immune SW), and immunizations were repeated 2 weeks later. Two weeks after the second vaccine, donor bone marrow cells (4 × 106) and spleen cells (10 × 106) were transplanted into C57BL/6 mice with lung micrometastases established by i.v. injection of 1 × 105 205-NP tumor cells (A)6 days prior to BMT. Recipients underwent 850 cGy TBI 1 day before BMT. One month after BMT, individual lung nodules were isolated from recipients, macerated, and placed in tissue culture. Seven lung nodule cultures were successfully grown from five recipients of Ag-Immune BMT,and 10 cultures were grown from four recipients of Non-Immune BMT. After culturing the cells for 2 weeks, they were stained with an unlabeled NP-specific antibody and a FITC-conjugated secondary antibody for analysis by flow cytometry. Background fluorescence was determined by staining of unmodified 205 tumor cells and also by staining of lung nodule cells with secondary antibody alone. Fluorescence histograms representative of the lung nodule cell cultures from Non-Immune recipients (B) and Ag-Immune recipients (C) are shown. Using the markers shown, the percentages of cells expressing NP were determined and plotted for each culture (D).
205-NP Lung nodules growing in recipients of Ag-Immune donor BMT exhibit loss of NP antigen expression. C3H.SW(SW) donors were immunized with 5 × 106 influenza virus-infected syngeneic spleen cells i.p.(Ag-Immune SW) or were not immunized (Non-Immune SW), and immunizations were repeated 2 weeks later. Two weeks after the second vaccine, donor bone marrow cells (4 × 106) and spleen cells (10 × 106) were transplanted into C57BL/6 mice with lung micrometastases established by i.v. injection of 1 × 105 205-NP tumor cells (A)6 days prior to BMT. Recipients underwent 850 cGy TBI 1 day before BMT. One month after BMT, individual lung nodules were isolated from recipients, macerated, and placed in tissue culture. Seven lung nodule cultures were successfully grown from five recipients of Ag-Immune BMT,and 10 cultures were grown from four recipients of Non-Immune BMT. After culturing the cells for 2 weeks, they were stained with an unlabeled NP-specific antibody and a FITC-conjugated secondary antibody for analysis by flow cytometry. Background fluorescence was determined by staining of unmodified 205 tumor cells and also by staining of lung nodule cells with secondary antibody alone. Fluorescence histograms representative of the lung nodule cell cultures from Non-Immune recipients (B) and Ag-Immune recipients (C) are shown. Using the markers shown, the percentages of cells expressing NP were determined and plotted for each culture (D).
Recipients of tumor antigen-immune BMT do not have an increased incidence of GVHD, even when exposed to whole tumor cell vaccines after BMT. C3H.SW (SW) donors were immunized with 5 × 106 influenza virus-infected syngeneic spleen cells i.p. (Ag-Immune SW) or 5 × 106 irradiated 205-NP tumor cells s.c.(Tumor-Immune SW), and immunizations were repeated 2 weeks later. Control donors were not immunized (Non-Immune SW). Two weeks after the second vaccine, donor bone marrow cells (4 × 106) and spleen cells(10 × 106) were transplanted into C57BL/6 mice given 850 cGy TBI 1 day before BMT. Recipients were not challenged with live tumor cells. A, whereas Tumor-Immune(allo-immune) control donor lymphocytes induced fatal GVHD in all recipients, NP Ag-Immune donor cells did not(n = 10). B, in an independent experiment, some recipients were exposed to NP antigen by s.c. injection of 5 × 106 irradiated 205-B7-NP tumor cells on days 0, 7, 14, and 21 after BMT to augment the ongoing immune response. Even in this case, there was no increase in the incidence of GVHD compared with recipients of Non-Immune donor BMT(n = 5).
Recipients of tumor antigen-immune BMT do not have an increased incidence of GVHD, even when exposed to whole tumor cell vaccines after BMT. C3H.SW (SW) donors were immunized with 5 × 106 influenza virus-infected syngeneic spleen cells i.p. (Ag-Immune SW) or 5 × 106 irradiated 205-NP tumor cells s.c.(Tumor-Immune SW), and immunizations were repeated 2 weeks later. Control donors were not immunized (Non-Immune SW). Two weeks after the second vaccine, donor bone marrow cells (4 × 106) and spleen cells(10 × 106) were transplanted into C57BL/6 mice given 850 cGy TBI 1 day before BMT. Recipients were not challenged with live tumor cells. A, whereas Tumor-Immune(allo-immune) control donor lymphocytes induced fatal GVHD in all recipients, NP Ag-Immune donor cells did not(n = 10). B, in an independent experiment, some recipients were exposed to NP antigen by s.c. injection of 5 × 106 irradiated 205-B7-NP tumor cells on days 0, 7, 14, and 21 after BMT to augment the ongoing immune response. Even in this case, there was no increase in the incidence of GVHD compared with recipients of Non-Immune donor BMT(n = 5).
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This work was supported in part by Clinical Oncology Career Development Award CDA-96-61 from the American Cancer Society (to C. A. M.), Research Project Grant RPG-98-035-01-CIM from the American Cancer Society (to C. A. M.), a grant from the Leukemia Research Foundation (to C. A. M.), and a Rosalie B. Hite Fellowship(to L. D. A.). Support for the Peptide Core Lab and veterinary services was provided by NIH Cancer Center Core Grant CA 16672.
The abbreviations used are: BMT, bone marrow transplantation; GVT, graft-versus-tumor; GVHD,graft-versus-host disease; NP, influenza nucleoprotein;mHAg, minor histocompatibility antigen; SW, C3H.SW; B6, C57BL/6; B7,B7.1; TBI, total body irradiation.