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CUTTING EDGE |
Department of Pathology and Department of Medicine, Section of Hematology/Oncology, University of Chicago, Chicago, IL 60637
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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-producing,
Th1/Tc1 phenotype may be optimal for tumor rejection. Recent work has
indicated that IFN signaling on tumor cells is important for protection
against carcinogenesis. However, the potential involvement of IFN
signaling among host immune cells has not been carefully examined. To
this end, Stat1-deficient mice were employed as tumor recipients. In
contrast to wild-type mice, Stat1-/- mice failed to
reject immunogenic tumors and did not support regression of poorly
immunogenic tumors when treated with an IL-12-based vaccine. T cells
from immunized Stat1-/- mice produced 50% of the levels
of IFN- and lacked cytolytic activity compared with wild-type mice,
and NK lytic activity also was not observed. Lack of cytolytic function
correlated with a failure to up-regulate serine esterase activity.
Thus, IFN-mediated signaling on host cells is required for the
development of antitumor lytic effector cells. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The role of IFN- itself in antitumor immunity is not entirely clear.
Mice deficient in IFN- signaling show increased susceptibility to
tumor induction by chemical carcinogens (6), and tumors
arising in those mice regain immunogenicity when IFN signaling is
reconstituted. Thus, one role played by IFN- is at the level of the
tumor cell, presumably by up-regulating the expression of MHC
molecules, Ag processing machinery, and perhaps tumor Ags as well.
However, a potential role for IFN signaling between host immune cells
in promoting antitumor T cell responses has not been carefully
examined.
Stat1-deficient mice have been generated by homologous recombination
and show a specific deficiency in all measured effects of signaling by
IFN-
ß and IFN-, with other cytokine-mediated responses
remaining intact (11, 12). An advantage of using
Stat1-deficient mice rather than IFN-R-deficient mice is the
elimination of a potential overlapping activity of IFN-ß
(13), some functions of which might compensate for the
absence of IFN- responsiveness. In the current study,
Stat1-deficient mice were examined for the ability to reject
immunogenic P815 variants and to control the growth of poorly
immunogenic P815 cells in response to IL-12-based vaccination.
Defective tumor rejection was observed, which correlated with a failure
to develop cytolytic T cells containing serine esterase activity. Thus,
in addition to a potential role at the effector phase of an antitumor
immune response, Stat1-dependent signaling can contribute to CTL
generation.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Stat1-deficient mice were generated as described previously (12) and generously provided by Dr. David Levy (New York University). These were backcrossed for five generations with DBA/2 mice (The Jackson Laboratory, Bar Harbor, ME). Heterozygous mice were intercrossed to obtain Stat1 wild-type (+/+), heterozygous (+/-), and homozygous knockout (-/-) mice. All mice were maintained under specific pathogen-free conditions in a barrier facility at the University of Chicago. Mice between 6 and 10 wk of age were used for experiments. PCR analysis of tail DNA was used to identify mice carrying the targeted Stat1 gene. Tail DNA was prepared using standard methods. A set of three primers was used for PCR analysis: P1 (5'-GAGATAATTCACAAAATCAGAGAG-3'), P2 (5'-CTGATCCAGGCAGGCGTTG-3'), and P3 (5'-TAATGTTTCATAGTTGGATATCAT-3'). Thirty-five cycles were performed, using an annealing temperature of 50°C. The PCR product was resolved using 1.5–2% agarose gels and visualized by ethidium bromide staining. Bands of distinct sizes corresponded to the wild-type and targeted alleles.
Cells
Three variants of P815 were used in this study: P1.HTR, a highly transfectable variant of P815 (14); P198, an immunogenic tum- clone of P815 (15);and P511, a subclone of P815 known to express the tumor Ag P1A. HTR.B7-1 and HTR.IL-12 were obtained by transfection of P1.HTR cells as described previously (16). The irrelevant syngeneic tumor L1210 and the NK-sensitive target YAC-1 were used as control targets.
In vivo tumor experiments
Cultured tumor cells were washed three times with DPBS, and 106 living cells were injected in 100 µl DPBS via a 27-gauge needle on the left flank. Tumor size was assessed twice per week using calipers, the longest and shortest diameters were measured, and a mean was calculated. Data from groups of three to five mice were analyzed at each time point, and a mean and SE were determined. Measurements were continued for 3–4 wk.
Immunization with irradiated P1.HTR transfectants
Cultured cells were washed three times in DPBS, adjusted to 108 cells/ml, and irradiated (10,000 cGy). Mice received 50 µl per hind footpad (5 x 106 cells) via a 27-gauge needle each week for 3 consecutive wk. In some experiments, naive mice were immunized, and, 2 wk after the last injection, splenocytes were stimulated in a mixed lymphocyte-tumor culture (MLTC)3 before assessing cytolytic activity. In other experiments, P1.HTR parental tumor cells were implanted first in the left flank of groups of mice, and, 1 wk later, immunization with irradiated P1.HTR transfectants was initiated with therapeutic intent.
Lymph node stimulation and lymphokine assays
Mice were injected into each hind footpad with
106 living HTR.IL-12 cells in 50 µl of DPBS;
control mice received DPBS. After 5 days, the draining popliteal lymph
nodes were harvested, and single cell suspensions were prepared. Cells
(106) were incubated in the presence or absence
of 2.5 x 105 irradiated (10,000 cGy)
HTR.B7-1 cells, supernatants were harvested after 48 h, and
residual cells were removed by centrifugation. IFN- and IL-4
concentrations were determined using an ELISA with Ab pairs obtained
from PharMingen (San Diego, CA). Concentrations were expressed in U/ml
or pg/ml as determined by the respective recombinant cytokines as
standards.
MLTC and cytolytic assay
Splenocytes (5 x 106) from immunized or control mice were stimulated with irradiated (10,000 cGy) HTR.B7-1 cells (2.5 x 105) in a volume of 2 ml, and 5–6 days later effector activities were analyzed. For cytolytic assays, unfractionated cells from the MLTC were washed, adjusted to 2 x 106/ml, and titrated in duplicate in V-bottom microtiter plates along with 2000 51Cr-labeled target cells. Supernatants were collected after 4 h and transferred to 96-well luma plates (Packard, Meriden, CT). After overnight incubation to allow drying, radioactivity was measured using a microplate scintillation counter (Packard). Percentage specific lysis was calculated using standard methods.
Serine esterase assay
Determination of total serine esterase activity was done by
measuring cellular content of
N--benzyloxycarbonyl-L-lysine-thiobenzyl
(BLT) esterase as previously described (17). Briefly, T
cells were purified by negative selection using a magnetic separation
system (StemCell Technologies, Vancouver, Canada) from MLTC cultures
generated as above. The purity of the eluted fraction, determined by
flow cytometry using anti-Thy-1 mAb, ranged between 95 and 97%.
Cells were centrifuged, washed three times in DMEM, and lysed in 200
µl of DPBS containing 1% Triton X-100; 100 µl of this lysate was
added in duplicate to 96-well plates. Assay solution (100 µl)
consisting of Tris buffer (pH 7.2) with 0.44 mM 5.5-dithio-bis(2
nitro)-benzoic acid and 0.40 mM BLT (Calbiochem, La Jolla, CA) was then
added. Plates were incubated (15–30 min) at 37°C, and absorbance was
measured at 405 nm using an ELISA plate reader.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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We previously reported that the spontaneous rejection of P198
cells by syngeneic DBA/2 mice was prevented by neutralization of
endogenous IL-12 and correlated with generation of T cells producing
high amounts of IFN- (3). To explore the role of
signaling by IFN among host cells, Stat1+/+,
Stat1+/-, and Stat1-/-
mice were challenged with immunogenic P198 cells, and tumor size was
measured over time. As shown in Fig. 1
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P198 was successfully rejected by Stat1+/+ and
Stat1+/- mice following an initial growth phase.
However, Stat1-/- mice failed to reject, with
all mice developing rapidly growing tumors and widespread
metastases, leading to death. These results indicate that IFN signaling
on host cells is necessary for the spontaneous rejection of this
immunogenic tumor.
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We previously reported that P1.HTR cells transfected to express
IL-12 (HTR.IL-12) were rejected in vivo, and that immunization with
irradiated HTR.IL-12 cells induced regression of preestablished P1.HTR
tumors (16). To investigate whether this rejection
depended on IFN signaling, P1.HTR tumors were implanted subcutaneously
in Stat1+/+ or Stat1-/-
DBA/2 mice. One week after tumor implantation, mice were treated weekly
with either irradiated HTR.IL-12 or with PBS. As shown in Fig. 2, immunization with irradiated HTR.IL-12
cells controlled tumor growth in Stat1+/+
but not in Stat1-/- mice. Similarly,
living HTR.IL-12 cells were rapidly rejected by wild-type mice but grew
progressively in Stat1-/- mice (data not
shown). Thus, efficacy of IL-12-facilitated antitumor immunity requires
IFN signaling on host cells.
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The lack of tumor rejection in Stat1-/-
mice could have been secondary to deficient T cell differentiation, or
due to a defect at the effector phase of the immune response via IFN
signaling on other cells. To examine whether a primed T cell phenotype
was generated in the absence of Stat1, HTR.IL-12 tumor cells were
injected in the hind footpads of Stat1+/+ or
Stat1-/- DBA/2 mice, and the draining popliteal
lymph node cells were harvested and restimulated to measure secreted
cytokines. As shown in Fig. 3, although T
cells from both wild-type and Stat1-deficient mice were primed to
produce IFN-, Stat1-/- T cells produced
approximately 50% of the level of IFN- seen with wild-type T cells.
Of note, there was not a corresponding increase in IL-4 production,
which was secreted only minimally by T cells from either group of mice
(Fig. 3B). In parallel experiments, a similar pattern of
IFN- production was observed following stimulation with anti-CD3
mAb, consistent with T cells being the source of the IFN- produced
(data not shown).
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production. Indeed,
in the above studies of T cells directly out of tumor-draining lymph
nodes, approximately 15% specific lysis was observed in wild-type
mice, but no specific lysis was detected from
Stat1-/- mice (data not shown). To elicit
higher levels of lytic activity for comparison,
Stat1+/+ or Stat1-/-
DBA/2 mice were immunized by three weekly injections of irradiated
HTR.IL-12 cells in the hind footpads. This regimen had been observed to
induce cytolytic activity previously (16). Splenocytes
from immunized or control mice were restimulated in vitro, and
cytolytic activity was measured against P815 and the NK target YAC-1.
As shown in Fig. 4A,
immunization with HTR.IL-12 generated P815-specific CTL activity, as
well as NK-mediated cytotoxicity, in Stat1+/+ but
not in Stat1-/- mice. Lysis against L1210
control cells was less than 10% in all cases, demonstrating
specificity (data not shown).
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B, T cells from
immunized Stat1+/+ showed increased serine
esterase activity compared with control T cells. In contrast,
up-regulation of serine esterase activity was not induced in
Stat1-deficient T cells. Collectively, these results indicate that IFN
signaling through Stat1 is required for the acquisition of a cytolytic
phenotype by antitumor T cells and NK cells in vivo. |
Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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-producing, Th1/Tc1-type T
cell response may be important for optimal rejection of established
tumors. In vivo, neutralization of B7/CD28 interactions or of IL-12
prevents CTL generation and blocks spontaneous tumor rejection
(2, 3). Conversely, coadministration of IL-12 augments the
efficacy of a variety of tumor Ag vaccines (7, 8, 20, 21),
and provision of IL-12 by other modalities also promotes antitumor
immunity (9, 16, 22). The fact that some biologic effects
of IL-12 can be blocked upon neutralization of IFN-
(23), and that tumor rejection has been observed to be
deficient in perforin knockout mice (5), have suggested
that the effector functions of a differentiated Tc1 phenotype are
important for tumor elimination. In fact, Tc1 cells have been shown to
be quantitatively superior to Tc2 cells as an adoptive immunotherapy
for established tumors (24). Our present results suggest
that IFN signaling among host cells is in fact necessary for the
development of antitumor cytolytic activity, revealing a surprisingly
critical role for IFN in peripheral T cell differentiation. A recent
report indicated that IL-4-deficient mice exhibited augmented CTL
activity (25), consistent with our present results.
Inasmuch as Stat1-/- mice are deficient in
signaling by IFN-ß as well as IFN-, either or both of these
cytokines might be important for CTL development. Both IFN- and
IFN- may contribute to T cell skewing toward a Th1-like phenotype
(13, 26, 27, 28). Moreover, IFN- is a potent inducer of NK
function and has a clear role in promoting anti-viral
CD8+ T cell responses (13, 29). On
the other hand, IFN- treatment has been reported to enhance IL-10
production by monocytes (30) and to inhibit IL-12
production by dendritic cells (31), both of which might be
expected to decrease Th1-like differentiation. Formal dissection of
whether signaling by IFN-ß or IFN- is required for CTL
generation will require additional experiments utilizing mice deficient
in IFN-R, IFN-ßR, or both.
The mechanism by which IFN signaling contributes to CTL maturation is not entirely clear. It seems likely that Stat1-/- T cells have an intrinsic defect in acquiring cytolytic activity. In support of this notion, preliminary experiments have indicated that immunization with tumor cells expressing B7-1, IL-12, and ICAM-1, which should replace the essential signals normally provided by professional APCs, still failed to induce specific CTL (data not shown). Stat1-dependent transcription may be vital for the expression of one of several genes encoding cytotoxic granule proteins. The observation that NK lytic activity also was defective supports the hypothesis of a generalized inability to develop cytolytic effector machinery in the absence of Stat1. However, an indirect role for Stat1 signaling via non-T cells in mediating this effect has not been excluded.
Previous work has indicated that IFN signaling at the level of the
tumor cell contributes to antitumor immunity (6). Human
lung cancer cells and melanoma cells deficient in IFN-R components
or Stat1 have been observed with high frequency (32).
However, our present data indicate an additional role for IFN signaling
on host cells for the development of effective antitumor immunity. A
recent report correlated the immunosuppressive effects of the
chemotherapy drug fludarabine with down-regulation of Stat1 signaling
in mononuclear cells (33). Collectively, these
observations suggest that defects in Stat1 activation by IFN treatment
of host lymphocytes should be explored as a potential mechanism of
immunosuppression in cancer-bearing patients.
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Acknowledgments |
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Footnotes |
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2 Address correspondence and reprint requests to: Dr. Thomas F. Gajewski, Department of Medicine, Section of Hematology/Oncology, MC2115, University of Chicago Medical Center, 5841 South Maryland Avenue, Chicago, IL 60637. E-mail address:
3 Abbreviations used in this paper: MLTC, mixed lymphocyte-tumor culture; BLT, N--benzyloxycarbonyl-L-lysine-thiobenzyl.
Received for publication July 14, 1999. Accepted for publication August 18, 1999.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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-dependent tumor surveillance system in immunocompetent mice. Proc. Natl. Acad. Sci. USA 95:7556./ß- and interleukin-12-mediated pathways in promoting T cell interferon responses during viral infection. J. Exp. Med. 189:1315. in mediating the antitumor efficacy of interleukin-12. J. Immunother. 17:71.
increases the frequency of interferon -producing human CD4+ T cells. J. Exp. Med. 178:1655.-interferon strongly up-regulates its own gene expression in peripheral blood lymphocytes. J. Exp. Med. 170:1021. in immune regulation. III. Differential selection of TH1 and TH2 murine helper T lymphocyte clones using recombinant IL-2 and recombinant IFN-. J. Immunol. 143:15.[Abstract]
stimulates production of interleukin-10 in activated CD4+ T cells and monocytes. Blood 87:4731.. J. Biol. Chem. 272:28779.This article has been cited by other articles:
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S. Ostrand-Rosenberg, M. J. Grusby, and V. K. Clements Cutting Edge: STAT6-Deficient Mice Have Enhanced Tumor Immunity to Primary and Metastatic Mammary Carcinoma J. Immunol., December 1, 2000; 165(11): 6015 - 6019. [Abstract] [Full Text] [PDF]
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A. K. Kacha, F. Fallarino, M. A. Markiewicz, and T. F. Gajewski Spontaneous Rejection of Poorly Immunogenic P1.HTR Tumors by Stat6-Deficient Mice J. Immunol., December 1, 2000; 165(11): 6024 - 6028. [Abstract] [Full Text] [PDF]
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P. Zhou, G. L. Szot, Z. Guo, O. Kim, G. He, J. Wang, M. J. Grusby, K. A. Newell, J. R. Thistlethwaite, J. A. Bluestone, et al. Role of STAT4 and STAT6 Signaling in Allograft Rejection and CTLA4-Ig-Mediated Tolerance J. Immunol., November 15, 2000; 165(10): 5580 - 5587. [Abstract] [Full Text] [PDF]
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C.-K. Lee, D. T. Rao, R. Gertner, R. Gimeno, A. B. Frey, and D. E. Levy Distinct Requirements for IFNs and STAT1 in NK Cell Function J. Immunol., October 1, 2000; 165(7): 3571 - 3577. [Abstract] [Full Text] [PDF]
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