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Immunology |
Departments of Experimental Pediatrics [L. D. A., D. P., L. A. E., C. A. M.] and Immunology [C. A. M.], The University of Texas M. D. Anderson Cancer Center, Houston, Texas 77030
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ABSTRACT |
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C3H.SW and C57BL/10 mice were immunized against a C57BL/6-derived fibrosarcoma or leukemia, and they were used as BMT donors. Recipients were H-2-matched, minor histocompatibility antigen-mismatched C57BL/6 mice with previously established micrometastatic tumors. Donor immunization led to a significant increase in GVT activity that was T cell dependent and cell dose dependent. In some settings, donor immunization also prolonged survival of recipients with preexisting micrometastatic tumors. However, donor immunization significantly increased the incidence of fatal graft-versus-host disease such that long-term survival was uncommon. In vitro cytotoxicity assays indicated that donor immunization induced both tumor-selective and alloreactive cytolytic T-cell populations. In vivo cross-protection assays showed that a substantial portion of the GVT effect was mediated by alloreactive cells not specific for the immunizing tumor. In conclusion, immunization of allogeneic BMT donors with a recipient-derived whole tumor cell vaccine substantially increases GVT activity but also exacerbates graft-versus-host disease.
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INTRODUCTION |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Allogeneic BMT3 is commonly used as a treatment for hematological malignancies and is under investigation as a treatment for several nonhematological tumors as well. In allogeneic BMT, donor lymphocytes that have not been tolerized by tumor and that have not been damaged by protracted chemotherapy are transferred to patients with minimal residual microscopic tumor burden. Such a setting may be conducive to successful cancer immunotherapy. Indeed, allogeneic BMT is associated with an unequivocal GVT effect, but the benefit of GVT activity is often offset by GVHD (7 , 8) .
It would be desirable to improve the therapeutic index of the immunological component of allogeneic BMT by causing the beneficial aspects of GVT activity to outweigh the toxicity of GVHD. In MHC-matched BMT, both tumor antigens and mHAgs remain largely undefined, and thus it is not clear to what extent the donor T-cell populations mediating GVT activity and GVHD are overlapping or identical (9 , 10) . Although both recipient tumor and GVHD target organs are "recipient self," tumors may have a different MHC/peptide epitope profile and thus may be capable of inducing different T-cell populations. Both clinical and experimental evidence suggests that some of the effector cells mediating GVT activity are distinct from those mediating GVHD (11, 12, 13, 14, 15) . For instance, even allogeneic BMT recipients that do not develop GVHD have a lower incidence of leukemia relapse compared with syngeneic BMT recipients or to recipients of T cell-depleted allogeneic BMT (11) . Furthermore, in vitro T-cell cloning and specificity studies have shown that some donor T cells that do not lyse normal recipient cells can lyse tumor targets (13, 14, 15) . GVT activity has also been demonstrated in F1 into parent murine BMT models that do not evoke GVHD, proving that the two responses can occur independently (12) .
In the course of a normal BMT, antigen-naive donor T cells are transferred to the tumor-bearing recipient, and development of GVHD represents a primary immune response to mHAgs. Target organs of GVHD all contain substantial numbers of professional antigen-presenting cells, which may efficiently stimulate primary immune responses to tissue-specific antigens (16 , 17) . However, primary immune responses to antigens on tumor cells in the recipient may not be induced efficiently, because tumor cells usually lack the necessary costimulatory molecules for efficient antigen presentation and generally do not have a large and organized population of antigen-presenting cells (18 , 19) . Therefore, conditions after transplant may favor development of a primary GVHD response that is greater than the primary GVT response.
For the reasons noted, it is conceivable that the relative potency of GVT activity could be increased by activating and expanding donor T-cell populations capable of recognizing tumor cells. One approach to this is immunization of donors with a recipient-derived tumor cell vaccine before donor cell harvest and transplantation. The experiments described in this study tested the hypothesis that immunization of normal immunocompetent MHC-matched donors with a recipient-derived tumor cell vaccine would substantially increase GVT activity and extend survival of BMT recipients with preexisting micrometastatic tumor. Antitumor activity and GVHD were assessed both in vivo and in vitro.
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MATERIALS AND METHODS |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Cell Lines.
205 is a weakly immunogenic, methylcholanthrene-induced C57BL/6 fibrosarcoma cell line (18)
. This tumor is not spontaneously metastatic but reproducibly forms multiple lung nodules when at least 1 x 104 cells are injected i.v. into C57BL/6 mice. 205 IL-2/TK is a 205 cell line modified to express both the IL-2 gene and the herpes simplex virus TK suicide gene using the LXSN and pBabePuro retroviral vectors, respectively. The pBabe Puro vector has a puromycin resistance gene (6)
, and the LXSN vector contains a neomycin resistance gene (20)
. Transduced cells were selected in 2.5 µg/ml puromycin and/or 1 mg/ml G418. B16F10 is a spontaneous, weakly immunogenic C57BL/6 melanoma cell line (a gift from Dr. I. J. Fidler, M. D. Anderson Cancer Center). When 105 cells are injected i.v. into C57BL/6 mice, multiple lung metastases are formed. C1498 is a C57BL/6 myelomonocytic leukemia cell line of spontaneous origin (American Type Culture Collection, Rockville, MD). 104 cells is a uniformly lethal dose of cells when injected i.v.; animals die with gross evidence of leukemia, including hepatomegaly and splenomegaly. Cells were grown in tissue culture using DMEM or RPMI 1640 supplemented with 5% heat-inactivated fetal bovine serum (Biowhittaker, Walkersville, MD), 100 units/ml penicillin, 100 µg/ml streptomycin, and 2 mML-glutamine.
BMT Donor Immunization.
Donors were immunized twice at 2-week intervals. Two to 3 weeks after the second immunization, mice were sacrificed for use as donors. For 205 tumor immunization, C3H.SW, C57BL/10, and C57BL/6 mice were injected s.c. in the flank with 3–5 x 106 205 IL-2/TK cells in 0.2 ml of HBSS on day 0, and they received 1 mg of ganciclovir i.p. in 0.2 ml PBS on days 3–9. Ganciclovir-mediated ablation of 205 IL-2/TK cells has been shown to induce systemic immunity to unmodified 205 tumor but is not cross-protective against B16F10 in C57BL/6 mice (21)
. Live 205 cells do not form tumors in C3H.SW mice. For alloimmunization, C3H.SW mice were injected s.c. in the flank with 20 x 106 C57BL/6 spleen cells in 0.2 ml HBSS. For C1498 tumor immunization, C3H.SW mice were injected s.c. in the flank with 3–7 x 106 30 Gy-irradiated C1498 leukemia cells in 0.2 ml of HBSS. Live C1498 cells do not grow progressively in C3H.SW mice.
In Vivo Tumor Inoculation.
Micrometastatic lung tumors were established by injecting C57BL/6 mice with 0.5–2 x 105 205 or B16F10 cells i.v. in 0.2 ml of HBSS 6 days before BMT (doses specified in text and legends). C1498 leukemia was established by injection of 1 x 105 cells i.v. 1 day before BMT (after TBI).
Bone Marrow Transplantation.
BMT recipients received 850 cGy TBI using a 60Co source 1 day before BMT. On the day of BMT, 2–4 x 106 bone marrow cells and 0–40 x 106 spleen cells were injected i.v. together in a total volume of 0.2 ml of HBSS. Bone marrow was isolated from donors by flushing each femur and tibia with RPMI 1640. Spleen cells were isolated by macerating the spleens between two frosted glass slides, followed by lysis of erythrocytes. Cell doses are specified in the text and legends. Early death from sepsis (in the first 2 weeks after BMT) was seen in some experiments in about 10% of recipients and was unrelated to treatment other than TBI. Such mice were excluded from analysis.
Enumeration of Pulmonary Tumor Nodules.
At the time of death or sacrifice, lungs from mice injected with 205 tumor 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. India ink was not used to count B16F10 nodules, which appear as dark brown nodules on unstained lungs.
Evaluation of GVHD.
Recipients were weighed weekly and observed for signs of GVHD (weight loss, alopecia, dermatitis, hunched posture, and death). In some experiments histological evidence corroborating the diagnosis of GVHD was obtained. Liver sections stained with H&E were examined for characteristic mononuclear infiltrates in portal triads.
Cytotoxicity Assays.
For use as effector cells, spleen cells were cultured in 6-well plates at 1 x 106 cells/ml and 10 ml/well in RPMI supplemented with 10% FBS (Summit Biotech, Ft. Collins, CO), 100 units/ml penicillin, 100 µg/ml streptomycin, 2 mML-glutamine, 100 mM sodium pyruvate, 0.1 mM nonessential amino acids, and 50 µM 2-mercaptoethanol (complete medium). In assays for alloreactivity, 30 Gy-irradiated C57BL/6 spleen cells were used as stimulators at a concentration of 1 x 106 cells/ml. In assays for combined tumor reactivity and alloreactivity, 250 Gy-irradiated 205 tumor cells were used as stimulators at a concentration of 5 x 103 cells/ml. After 4–5 days in culture with the appropriate stimulator cells, effector cells were harvested and plated in triplicate with 5 x 103 51Cr-labeled target cells per well at E:T ratios ranging from 300:1 to 19:1. Target cells were labeled by combining 5 x 106 cells in 0.1 ml of complete medium with 20 µl FBS (Summit) and 0.1 ml (
100 µCi) sterile isotonic Na251CrO4 (Amersham, Arlington Heights, IL) for 60 min at 37°C. Con A lymphoblast targets were generated by stimulating C57BL/6 spleen cells for 2 days with 2 µg/ml Con A at 2 x 106 cells/ml in complete medium. They were labeled with 51Cr as above for 45 min. 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 x [(experimental cpm - spontaneous cpm)/(maximum cpm - spontaneous cpm)]. Spontaneous release was usually <20% and always <30% of the maximum release.
Limiting Dilution Analysis.
To determine the precursor frequency of alloreactive and tumor-reactive cytolytic lymphocytes, graded numbers of spleen cells were cultured in replicates of 24–40 in 96-well, round-bottomed plates along with 250 Gy-irradiated 205 stimulator cells (500/well) or 30 Gy-irradiated C57BL/6 spleen cell stimulators (5 x 104/well) in 0.2 ml. Cultures were fed on days 4 and 7 by replacing 0.1 ml of supernatant with fresh complete medium containing IL-2 (final concentration, 5 IU/ml; Chiron, Emmoryville, CA). On day 11, 5 x 103 target cells (51Cr-labeled C57BL/6 Con A lymphoblasts or 205 tumor cells) were added to each well, and cytotoxicity was measured as above. CTL precursor frequency was estimated based on the Poisson distribution and probability theory, which predicts that an average of one CTL precursor per well will result in the absence of lysis in 37% of the wells, as described (22
, 23)
. Lysis was defined as 3 SD above the mean spontaneous 51Cr release.
Split-Well Specificity Assay.
To determine the target specificity of cytolytic T cells, donor spleen cells were cultured using limiting dilution conditions as above. On day 11, the cells from each well were transferred in equal portions to two new wells and tested in parallel against two different 51Cr-labeled targets. Cytotoxicity was measured as above, and data were analyzed to determine whether particular clonal populations lysed both targets or only one of the targets.
T-Cell Subset Depletion.
Prior to BMT, immune donor spleen cells were incubated on ice for 30 min with mAb to either CD4 (clone GK1.5; PharMingen, San Diego, CA) or CD8 (clone 53-6.72; PharMingen) using a 1:100 dilution of antibody and a cell concentration of 60 x 106 cells/ml. They were then washed twice and incubated at 4°C for 15 min with a polyclonal F(ab')2 secondary goat anti-rat IgG antibody coupled to a paramagnetic microbead (Miltenyi Biotec, Auburn, CA). The cells were next passed twice through a negative selection column using a SuperMACS separator (Miltenyi Biotec.). The cell population that did not bind to the column contained essentially either no CD4+ cells or no CD8+ cells, as measured by flow cytometry (data not shown). As a control for BMT, some immune donor spleen cells were treated with labeled secondary antibody only and passed through a negative selection column in the same manner as the depleted cells (sham-depletion). After T-cell subset depletion, 4 x 106 processed spleen cells were injected into BMT recipients along with 4 x 106 bone marrow cells.
In other experiments, antibodies and complement were used to deplete cells. Immune donor spleen cells were incubated with monoclonal antibody to either CD4 (clone GK1.5) or CD8 (clone 116-13.1, a gift from Dr. M. Kripke, M. D. Anderson Cancer Center) as above. They were then washed twice and incubated at 37°C for 45 min with a 1:16 dilution of low-toxicity baby rabbit complement (Serotec, Raleigh, NC). The cells were again washed twice, and the entire procedure was repeated. CD4+ and CD8+ cells were depleted to background levels as measured by flow cytometry (data not shown). Processed cells (10 x 106) were injected into BMT recipients along with 4 x 106 bone marrow cells.
Statistical Analysis.
Prism 2.01 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 survival curves, the log-rank test was used. Significant results were verified using a Cox-Mantel test using the statistical software Statistica 5.1 (StatSoft, Tulsa OK). To compare numbers of tumor-free mice, a Fisher’s Exact test was used.
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RESULTS |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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). Furthermore, 8 of 10 recipients of immune allogeneic donor cells and 5 of 5 recipients of immune syngeneic donor cells were completely free of lung nodules, whereas 0 of 14 recipients of naive donor cells were tumor free (P < 0.05 for each strain). This GVT activity from 205-immune C3H.SW spleen cells was T cell mediated; it was abolished by in vitro depletion of CD8+ cells and reduced by depletion of CD4+ cells in the graft before BMT (data not shown).
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), and doses of 2, 5, and 10 x 106 spleen cells each afforded complete protection against growth of metastatic 205 pulmonary nodules.
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Tumor-bearing BMT Recipients Exhibit Prolonged Survival after BMT Using Tumor-immune Allogeneic Donors.
Because tumor-immune donors have both alloreactive and tumor-reactive T cells in their repertoire, experiments were conducted in vivo to compare the relative magnitude of these activities. In the dose-response experiment described above, survival was slightly increased at all doses of 205-immune donor spleen cells compared with the group that received only bone marrow, and the increase was statistically significant at the dose of 1 x 106 spleen cells (mean, 39 ± 2 days versus 23 ± 2 days for the control group, P < 0.01; Fig. 1B
).
This observation was extended in an experiment in which BMT recipients received a minimum tumor-curative dose of immune C3H.SW donor spleen cells (2 x 106) or an equal dose of naive C3H.SW donor cells. In the group receiving immune donor cells, survival was significantly increased (P < 0.05; Fig. 2A
), and the number of lung tumor nodules was significantly reduced (mean, 0.4 ± 0.2 compared with 50 ± 8 for controls, P = 0.0002; Fig. 2B
). However, all recipients of immune donor cells ultimately died with GVHD.
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). Although four of five recipients died with signs of both acute GVHD and leukemia, one recipient survived >100 days without evidence of leukemia or GVHD.
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. However, 10 x 106 205-immune cells were nearly uniformly lethal, and as few as 1 x 106 205-immune spleen cells also induced substantial mortality. A similar result was seen using C1498-immune donors, which induced fatal GVHD in 40–60% of recipients. These results indicated that tumor immunization increased the number of alloreactive lymphocytes in C3H.SW donors. Consistent with this was the 2.3–6.5-fold increase in CTL precursors capable of lysing C57BL/6 Con A lymphoblasts observed in limiting dilution assays (data not shown). Approximately 40% of the recipients of tumor-immune cells survived beyond 100 days. Many exhibited temporary weight loss and dermatological changes characteristic of acute GVHD before they resumed weight gain (data not shown).
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). In syngeneic C57BL/6 mice, 205 immunization prevents the growth of 205 but not B16F10 (21)
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DISCUSSION |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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The simplest explanation of these findings is that donor immunization activated and expanded T-cell clones that recognized recipient mHAgs expressed on tumor cells and normal recipient tissues. According to this interpretation, GVT activity is simply another manifestation of GVHD. This is consistent with the observations that: (a) donor immunization with 205 tumor cells also protected against the syngeneic but immunologically unrelated B16F10 tumor; (b) alloimmunization with C57BL/6 spleen cells produced substantial protection against both tumors; and (c) increased GVHD mortality was seen in recipients that had never been challenged with tumor. Allogeneic donor cells have been shown to mediate GVT activity against an MHC-mismatched tumor (26) . An alternative interpretation is that two populations of T cells were activated: one reactive with antigens predominantly found on tumor, and the other reactive with ubiquitously distributed mHAgs. The facts cited above provide unambiguous evidence for the second population. Induction of a tumor-selective population is compatible with the observations that: (a) under limiting dilution conditions, many tumor-reactive donor T cells did not lyse other C57BL/6 targets; and (b) antitumor activity was seen after BMT using tumor-immune C57BL/6 syngeneic donors. However, in allogeneic BMT experiments in vivo, we saw no compelling evidence of a tumor-specific effect.
These experiments show that although donor immunization with tumor cells induces powerful GVT activity, whole tumor cells are too complex to be used safely for donor sensitization strategies because of the unpredictable and uncharacterized nature of mHAg expression by tumors. Any method of stimulating allogeneic donor T cells with a patient’s tumor cells before BMT may lead to an increased risk of inducing severe GVHD in the patient. Although in some patients and models GVT activity may be separated from GVHD by depletion of CD8+ T cells (27 , 28) , such depletion was shown to abolish the GVT activity in this study.
There is substantial interest in finding methods that will dissociate GVT activity and GVHD in allogeneic BMT. Underlying many of these efforts is the assumption that tumor cells have some unique (or at least not widely expressed) immunogenic antigens. In some human malignancies, such as melanoma, antigens with relatively restricted tissue distribution have been identified (29 , 30) . Although our results show that immunization with complex allogeneic tumor vaccines induces GVHD, immunization of allogeneic donors with defined tumor antigens or immunodominant peptide fragments may selectively increase GVT activity. Preliminary studies in our laboratory show that donor immunization with a model tumor antigen does not exacerbate GVHD.
It is possible that in most cases there are no unique tumor targets for GVT activity or that any unique tumor antigens are overshadowed by immunodominant mHAgs. In this case, induction of GVHD may still have potential as an anticancer therapy. It may need to be used in a manner similar to chemotherapy, with careful titration of cell doses and immunosuppressive therapy, and application at times in which tumor cells are maximally sensitive to GVHD and critical organs are minimally sensitive to GVHD.
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FOOTNOTES |
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1 This work was supported in part by a Clinical Oncology Career Development Award (CDA-96-61) from the American Cancer Society (to C. A. M.), a Research Project Grant (RPG-98-035-01-C1M) from the American Cancer Society (to C. A. M.), a grant from the Leukemia Research Foundation (to C. A. M.), and a predoctoral fellowship from the Rosalie B. Hite Fellowship Committee of M. D. Anderson Cancer Center (to L. D. A.). 
2 To whom requests for reprints should be addressed, at Department of Experimental Pediatrics, The University of Texas M. D. Anderson Cancer Center, Box 88, Room B7.4518, 1515 Holcombe Boulevard, Houston, TX 77030. Phone: (713) 792-3314; Fax: (713) 794-4373; E-mail: mullen{at}mdacc.tmc.edu
3 The abbreviations used are: BMT, bone marrow transplantation; GVT, graft versus tumor; GVHD, graft-versus-host disease; mHAg, minor histocompatibility antigen; TK, thymidine kinase; IL, interleukin; TBI, total body irradiation; Con A, concanavalin A.
Received 10/12/98. Accepted 1/29/99.
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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, P < 0.01 compared with control recipients of "0 spleen cells."
75:1 compared with lysis by naive SW effectors.