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Immunology |
Department of Microbiology and the University of Pennsylvania Cancer Center, University of Pennsylvania, Philadelphia, Pennsylvania 19104-6076
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ABSTRACT |
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 Top ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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INTRODUCTION |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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B16F10 melanoma is among the most aggressive, poorly immunogenic murine tumors (4) . In addition, B16F10-NP tumor cells lose expression of the NP antigen transgene very rapidly with the emergence of antigen loss tumor cells in vivo (3) . Thus, among transplantable mouse tumors, this model tumor system provides a stringent test of the potency of any immunotherapeutic approach. In this study, therefore, we have attempted to optimize a parenteral vaccine protocol using Lm-NP to determine whether conditions exist under which we can cure mice of established s.c. tumors and of experimental lung "metastases" established by tail-vein injection with B16F10-NP cells.
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MATERIALS AND METHODS |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Cell Lines.
B16F10 is a spontaneously arising melanoma cell line derived from the C57BL/6 mouse that shows very low expression of MHC class I and no expression of MHC class II (4)
. It was transduced with a NP gene (A/PR8/34) using a replication-defective Moloney murine leukemia retrovirus containing both the NP gene and a neomycin phosphotransferase gene, as described previously (5
, 6) . Transductants were selected in G418 and retain the NP gene in vitro in the presence of G418. The expression of NP in this tumor does not enhance its immunogenicity or decrease its intrinsic tumorogenicity in that the minimal tumoricidal dose in mice is identical to the parent line; for B16F10-NP, 2 x 103/mouse. Thus, when expressed in the tumor, NP behaves indistinguishably from an endogenous tumor antigen.
B16F10-GM-CSF is B16F10 transduced with murine GM-CSF, as described previously (7) . It was kindly provided by Drew Pardoll (Johns Hopkins School of Medicine, Baltimore, MD).
Cells were maintained in RPMI 1640 containing 10% FCS, 2 mM L-glutamine, 50 units/ml penicillin, and 50 µg/ml streptomycin.
L. monocytogenes Strains and Propagation.
The L. monocytogenes vaccine strain used in this study is DP-L2028, which was constructed from the hemolytic wild-type strain 10403S to stably express and secrete a fusion protein of listeriolysin, an essential virulence factor of the bacterium and influenza (A/PR8/34) nucleoprotein, LLO-NP. The construction of the L. monocytogenes recombinant DP-L2028 (Lm-NP) has been described previously (8)
. In some experiments, as a control for nonspecific effects mediated by the bacterial vector alone, we used a stable chromosomal recombinant of L. monocytogenes that synthesizes and secretes the HIV Gag protein (9)
. In this study, we refer to these strains as Lm-Gag and Lm-NP to draw attention to their antigenic difference. Lm-Gag has a similar LD50 in C57BL/6 mice to that of Lm-NP (
4 x 108 CFU; Ref. 8
). Both strains have attenuated virulence, of about three logs, compared with the parental strain 10403s from which they were constructed. The bacteria are propagated in brain-heart infusion medium broth and agar. Lm-NP is maintained in medium supplemented with 25 µg/ml of chloramphenicol to select for the LLO-NP-containing plasmid.
Vaccinations.
Lm-NP and Lm-Gag were administered i.p. using the doses described. Vaccination by vaccinia (107 PFU) was via the tail vein. Vaccinia expressing the NP gene of PR8 was kindly provided by Dr. Jack Bennink (Laboratory of Viral Diseases, National Institute of Allergy and Infectious Disease). Vaccinia expressing the E7 gene of human papilloma virus strain 16 was kindly provided by Dr. T-C. Wu (Johns Hopkins University, Baltimore, MD). B16F10-GM-CSF immunizations were performed with a total of 1.5 x 107 irradiated (6000 rads) cells. Mice received 3 x 106 B16F10-GM-CSF tumor cells in each limb, plus the back.
Tumor Regression Studies.
B16F10-NP (2 or 3 x 105) was introduced s.c. into C57BL/6 mice. After measurable (4–5-mm) tumors had grown (between 7 and 11 days after tumor challenge), treatment was initiated. Groups of 8 or 10 mice were used for each treatment, and all animals were randomized before receiving treatment. Tumor growth was then measured using calipers every 2 days and was recorded as the narrowest and longest surface length. Values shown for tumor size (mm) are the mean of these two lengths for each animal in the group. Mice were sacrificed when tumor sizes reached 20–25 mm in average diameter. Statistical significance was determined by t test (Statworks, version 1.2). In all experiments, a P 
0.05 was considered significant. Each experiment was performed at least twice with essentially similar results, unless otherwise described.
Treatment of B16F10-NP Pulmonary Metastases.
Fifty mice were injected with 4 x 104 B16F10-NP tumor cells in the tail vein to establish pulmonary metastases. All animals were randomized before receiving treatment and divided into five groups of 10 mice. As a control, one group of 10 mice was left untreated. Two groups were immunized with 4 x 106 CFU, 107 CFU, and 4 x 107 CFU of Lm-NP on either day 6, 12, and 18 or day 9, 15, and 21 after i.v. challenge with tumor cells. The other two groups received identical doses of Lm-Gag on a similar schedule. The animals were sacrificed on day 50, and lungs from the mice were inflated with PBS and then fixed in 10% buffered formalin phosphate. Metastases were counted under a dissecting microscope.
Analysis of Cytokine mRNA Expression in B16F10-NP Tumors by RT-PCR.
Mice were injected s.c. with 2 x 105 B16F10-NP. At a time when the tumors reached 5 mm in size, the mice were immunized i.p. with 2 x 106 CFU Lm-NP or Lm-Gag; seven days later, a second dose of Lm-NP was administered. One, 3, or 7 days after the second immunization, tumors were harvested and frozen immediately in liquid nitrogen for RT-PCR analysis. The tissue was then homogenized and lysed in a guanidinium-based solution for purification of total RNA using the RNeasy total RNA kit (QIAGEN), according to the manufacturer’s directions. For first-strand cDNA synthesis the SuperScript kit (Life Technologies, Inc.) was used. For amplification of the target cDNA, PCR primers were designed to encompass a length of 300 bp of cDNA. Primer sequences are described in Table 1
and have been shown competent to amplify the appropriate gene product. After PCR amplification, the cDNA samples were run on a 0.9% agarose gel at 100 V for 1–2 h with ethidium bromide staining and compared with a 123-bp ladder marker to detect cDNA products of appropriate size. 
-Actin levels were used to standardize cDNA template concentrations used in PCR reactions. Representative results are presented from a single set of experiments; the isolation of RNA from the various groups of tumor-bearing mice was performed three times, and each set of RNA isolates was analyzed by RT-PCR at least three times.
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RESULTS |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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. The animals that were cured of established B16F10-NP tumor resisted further challenge with the tumor given on day 30. In an attempt to improve this cure rate, we investigated the effects of repeated immunization at weekly intervals with the Lm-NP vaccine, which may provide long-term activation of anti-NP CTL (10)
. In the first experiment, B16F10-NP tumors were established in eight mice and immunization began at day 7; twenty-five percent of the mice were cured by a single immunization, and an additional 25% were cured after a second immunization on day 14. After the third immunization on day 21, the tumor was eliminated in one other mouse and tumor growth was arrested in an additional mouse. No additional cures were observed after the fourth immunization on day 28 or the fifth immunization on day 35, although tumor growth was halted in all but one mouse so that at 6 weeks after tumor challenge seven of eight mice in the Lm-NP-immunized mice survived, whereas all of the mice in the other groups had been euthanized (Fig. 1B)
. In a second experiment, five mice in each group were immunized weekly for 7 weeks. After five immunizations, a total of 60% of mice were cured by this protocol and remained tumor free until we terminated the experiment at around 3 months after tumor challenge (data not shown). No further cures were effected by the last two immunizations in this experiment. Thus, repeated immunization with 2 x 106 CFU of Lm-NP improves the cure rate of established B16F10-NP tumors slightly and also inhibits tumor growth in nonregressing tumor-bearing mice.
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shows the effect on regression of B16F10-NP by delivering Lm-NP or Lm-Gag on day 7 (4 x 106 CFU), day 14 (107 CFU), and day 21 (2 x 107 CFU). This did not increase the cure rate of B16F10-NP to greater than that achieved using lower doses (Fig. 1, A, B, and C)
. We conclude that repeated immunizations of small doses are more effective than increased doses of the L. monocytogenes vaccine.
In the experiments shown in Fig. 1, B and C
, we included a control for nonspecific immunity. We had not observed any slowing of tumor growth due to the i.p. delivery of 2 x 106 CFU of vector alone with RENCA-NP and CT-26-NP (2)
or 108 to 109 PFU p.o. with RENCA, CT-26, or B16F10-NP (3)
. However, because the dose was repeated (Fig. 1B)
or escalated (Fig. 1C)
for the subsequent immunizations we included Lm-Gag, a strain of L. monocytogenes that synthesizes and secretes the HIV Gag protein, which has similar virulence to Lm-NP. In Fig. 1, B and C
, we see a slowing of tumor growth in animals who received Lm-Gag compared with naive mice that by day 14 and later time points in the experiments shown in Fig. 1, B and C
, was statistically significant (on day 14: Fig. 1B
, P > 0.015 for Lm-Gag relative to naive mice compared with P > 0.0001 for Lm-NP relative to naive mice; Fig. 1C
, P > 0.005 for Lm-Gag relative to naive mice compared with P > 0.0001 for Lm-NP relative to naive mice). Although Lm-Gag clearly exercises a potent adjuvant effect on tumor growth, we have never observed long-term survivors in this treatment group of mice, in contrast to mice cured of established tumors by Lm-NP that typically remain tumor free for several months and resist subsequent tumor challenges.
Combined Therapy with L. monocytogenes-NP and Vaccinia-NP Does Not Improve the Cure Rate of B16F10-NP Tumors.
Another approach to eliminating the limits imposed by pre-exposure to the L. monocytogenes vector is to use a combination therapy that delivers the tumor antigen with a different vector. Vaccinia virus that expresses the same NP gene as Lm-NP does not impact greatly on the growth of established B16F10-NP. In Fig. 2A
, only one of eight mice is cured by multiple immunizations of vaccinia-NP. Nevertheless, a booster immunization using a different NP-expressing vector might preferentially activate NP-specific, rather than L. monocytogenes-specific, T cells. We, thus, used Lm-NP for primary immunization and vaccinia-NP for subsequent boosters (Fig. 2B)
. This adjunctive therapy did not improve the best cure rate that we obtained using Lm-NP alone (Fig. 2B
compared with Fig. 1
).
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shows, five of eight animals that received both Lm-NP plus irradiated B16F10-GM-CSF became tumor free, four of eight mice that received Lm-NP became tumor free, and three of eight mice that received irradiated B16F10-GM-CSF alone became tumor free. On day 35, the average tumor size for naive animals was 23.6 ± 2.16 compared with 10.8 ± 11.74 for B16-GM-CSF-immunized mice, 4.3 ± 4.65 for Lm-NP-immunized mice, and 1.8 ± 2.43 for mice that received the combined therapy. The difference between the groups of animals that received Lm-NP or Lm-NP and B16F10-GM-CSF combined was not statistically significant (P = 0.16), nor was the difference between animals that received Lm-NP or B16F10-GM-CSF (P = 0.15). However, the difference between the animals that received combined therapy or B16F10-GM-CSF was marginally significant (P = 0.04). Thus, the tumors progressed more rapidly in mice treated with irradiated B16F10-GM-CSF alone, and the tumors in the mice that received the combined therapy grew the slowest (Fig. 2b)
. We repeated this experiment including a group of mice that received Lm-NP plus irradiated B16F10 tumor cells that did not express GM-CSF. We found no statistical difference between the numbers of mice that became tumor free in the groups of mice that received Lm-NP alone, Lm-NP plus B16F10-GM-CSF, or Lm-NP plus B16F10. In all groups, 35–50% of the mice showed regression of B16F10 tumors of about 2 mm (data not shown). Taken together, these data suggest that a combined therapy of Lm-NP with irradiated B16F10-GM-CSF did not greatly improve the Lm-NP single therapy.
GM-CSF Is Found in B16F10-NP Tumors from Mice Immunized with Lm-NP.
The lack of synergy between Lm-NP and B16F10-GM-CSF suggests that GM-CSF is not a limiting factor in Lm-NP therapy. These data also suggest a lack of synergy between the anti-NP response and the antimelanoma antigen response facilitated by GM-CSF. Conceivably, cellular infiltrates into necrotizing B16F10-NP tumors could be secreting GM-CSF after treatment with Lm-NP. To test this hypothesis, we examined whether GM-CSF was, indeed, found in tumors for mice immunized with Lm-NP using RT-PCR. Fig. 3
shows that GM-CSF is detected in B16F10-NP at day 7 after immunization with Lm-NP, whereas GM-CSF is not detected after treatment with Lm-Gag.
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Mice received two immunizations 6 days apart on days 12 and 18 or days 15 and 21 after i.v. challenge. Metastases were counted on day 50 after tumor challenge. Table 2
shows that 5 of 10 mice who received Lm-NP on days 6, 12, and 18 were metastases free and 3 of 10 mice who received Lm-NP on days 9, 15, and 21 were metastases free. Although there is a strong nonspecific slowing of tumor growth in Lm-Gag-immunized mice, only 1 of 10 mice who received Lm-Gag on days 6, 12, and 18 were free of metastases and none of the mice who received Lm-Gag on days 9,15, and 21 were tumor free. Clearly, Lm-NP is as effective at treating micrometastatic disease as it is at treating established primary tumors.
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and Table 2
) indicates a nonantigen-specific component of therapy with live L. monocytogenes vaccines. Although Lm-Gag only slows tumor progression and does not cure mice with established tumors, infection with L. monocytogenes clearly results in the production of soluble mediators that either directly kill B16F10-NP or slows their growth rate. L. monocytogenes infections are associated with strong Th1 CD4+ T cell responses, including the production of IL-12, IFN-
, and TNF (1)
. We, thus, examined the profile of mRNA expression for IL-1, IL-2, IL-6, IL-12 p40; IFN-
, -, and -; TNF- and - in B16F10-NP tumors from mice immunized with Lm-NP versus Lm-Gag using RT-PCR. Fig. 3
shows the up-regulation of mRNA for all of these cytokines, except IFN- and TNF- in tumor-bearing mice treated with Lm-NP at days 1, 3, and/or 7 after immunization. However, tumors from mice immunized with Lm-Gag also showed elevated levels of mRNA for most of these cytokines compared with tumors grown in unimmunized animals, suggesting that they could play a role in slowing tumor growth. Curiously, IFN- and TNF-, which are the hallmark of a listerial infection, were undetectable in the tumors at these time points (data not shown). It is possible, however, that they are expressed in the tumor nodule at later time points than day 7, the last time point evaluated. |
DISCUSSION |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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In the present study, we have extended our findings that an L. monocytogenes vector can cure mice of established tumors to a more tumorigenic model, that of B16F10. We show that we can effectively and permanently cure 50–60% of syngeneic C57BL/6 mice of palpable primary s.c. tumors and established experimental lung metastases using LM-NP alone as the therapy and that this is just as effective as combination therapies with recombinant vaccinia vectors or GM-CSF-complemented autologous irradiated tumor cells. The lack of synergy between B16F10-GM-CSF-induced immunity and Lm-NP was unexpected. GM-CSF-based tumor immunotherapy is directed at inducing cell-mediated immunity to endogenous tumor antigens (7)
. It has recently been shown that the induction of this immunity is crucially dependent on CD4+ T cells of both the Th1 and Th2 phenotype in that B16F10-GM-CSF-primed IFN--/- and IL-4-/- mice are impaired in their ability to reject B16F10 tumors (18)
. Th2 cells are thought to induce eosinophils that are abundant at the site of tumor challenge (18)
. It is possible that the strong Th1 immunity induced by Lm-NP impairs the ability of B16F10-GM-CSF to induce appropriate CD4+ effector cells. On the other hand, the presence of mRNA for GM-CSF in regressing tumors from Lm-NP immunized mice indicates that this cytokine is present in tumors after Lm-NP infection. The potent cytokine response induced by treatment of tumor-bearing mice with Lm-NP could conceivably function to activate T cell-immunity against endogenous melanoma antigens, in addition to NP, in the absence of the irradiated B16F10-GM-CSF vaccine.
These studies provide the first demonstration that, in addition to inducing potent antigen-specific therapy, L. monocytogenes has an adjuvant effect that results in significant nonspecific slowing of tumor growth. This correlates with expression of mRNA for IL-1, IL-2, IL-6, IL-12, IFN- and -, and TNF- in tumors treated with Lm-vaccines. There are a number of ways in which cytokines produced by L. monocytogenes infection may directly interfere with tumor growth. IFN- in combination with IL-1 has been shown to inhibit the growth of B16F10 by a mechanism that does not involve a direct inhibition of tumor cell growth (19)
. The IFNs are known to increase MHC (20)
and Transporters associated with Antigen Processing (21)
expression in human melanomas. B16F10 is notoriously low in MHC class I expression; in fact the cell line we use is Kb negative and expresses only low levels of Db. We find that B16F10-NP explanted from mice immunized with Lm-NP has up-regulated Kb expression, as measured by fluorescence-activated cell-sorting analysis, to significant levels (MFI background, 2–10 x 102) and increased Db expression 10-fold (MFI background of in vitro cultured tumors, 2 x 102; MFI background in explanted tumors, 1.5 to 2 x 103). Up-regulation of MHC class I expression of B16F10 in mice that have been immunized with Lm-Gag could result in the presentation of otherwise silent endogenous melanoma antigens. In addition, IL-12 has been shown to be a potent inhibitor of angiogenesis in the B16F10 tumor model, in addition to possibly activating and recruiting antitumor lymphocytes (22)
. IL-6 may recruit neutrophils and other phagocytic cells into the tumor milieu, whereas TNF- can synergize with other cytokines in direct killing of tumor cells. The nonspecific activation of inflammatory cytokines by intracellular bacteria such as Bacillus Calmette-Guérin has been used for decades in cancer therapy (23
, 24)
. Recently Salmonella typhimurium has also been introduced into the armory of antitumor agents (25)
. The potency of L. monocytogenes as an anticancer immunotherapeutic may be the result of the combination of the adjuvant property of this bacterium and its ability to induce potent antigen-specific cell-mediated immunity. The contribution of the adjuvant effects of the vector vehicle to antitumor immunity induced by the vectored antigen clearly warrants further investigation.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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1 Supported by NIH Grant CA69632 (to Y. P.). L. M. W. is supported by training Grant 2T32CA09140. 
2 To whom requests for reprints should be addressed, at Department of Microbiology, University of Pennsylvania, 3610 Hamilton Walk, 323 Johnson Pavilion, Philadelphia, PA 19104-6076.
3 The abbreviations used are: NP, influenza nucleoprotein; GM-CSF, granulocyte macrophage colony-stimulating factor; CFU, colony-forming unit; RT-PCR, reverse transcription-PCR; MFI, mean fluorescence intensity; IL, interleukin; TNF, tumor necrosis factor.
Received 6/14/99. Accepted 8/20/99.
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