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Departments of Microbiology and Pathology, Saskatoon Cancer Center, Saskatoon, Saskatchewan, Canada;
Department of Veterinary Microbiology, Western College of Veterinary Medicine, Saskatoon, Saskatchewan, Canada;
Veterinary Infectious Disease Organization, University of Saskatchewan, Saskatoon, Saskatchewan, Canada; and
Department of Pathology, University of Ottawa, Ottawa, Ontario, Canada
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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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, IFN-
, or GM-CSF) can
induce tumor rejection responses (1, 2, 3). Multiple effector
mechanisms apparently mediate these responses (1, 4), but
most reportedly depend on the actions of cytotoxic T cells
(1, 2, 3). Whereas vaccination before tumor introduction or
shortly thereafter can induce effective rejection responses,
therapeutic vaccination at later stages of tumor growth generally fails
(5). This failure is not due to systemic T cell exhaustion
or systemic anergy, because tumor-bearing mice can still reject tumor
cells engineered to secrete cytokines, and even late vaccination of
tumor-bearing hosts can induce specific immune responses that mediate
rejection of subsequent tumor cell challenges (6).
Therefore, the failure of cancer vaccines to mediate rejection of
established tumors could be at least in part the result of relatively
lesser degrees of T cell infiltration into, or activation within,
tumors and/or the growth of the tumor cell population outpacing that of
the specific T cells.
The trafficking of lymphocytes into tissues is a dynamic, multistep
process. It involves both intra- and extravascular processes and
ultimately, in the case of tissue reactions, migration of the
lymphocytes along chemoattractant gradients (7, 8)
established by chemokines produced at the site of inflammation
(9). Chemokines are a superfamily of chemoattractant
cytokines (10) produced by multiple cell types (e.g.,
leukocytes, endothelial cells, tumor cells) in response to an array of
inflammatory stimuli (11). They are related genetically by
a conserved four-cysteine motif, with the three major families of the
superfamily defined by the spacing of the first two cysteines in this
conserved motif. For the family, the two cysteines are separated by
any residue (CXC), whereas the analogous two cysteines of the 
family are adjacent (CC), and the newly identified family (C)
possesses only one cysteine at its N terminus (12).
Generally, the CXC chemokines are potent activators and
chemoattractants for neutrophils, whereas the CC and C chemokines have
the potential to chemoattract monocytes and T lymphocytes
(12). The T cell specificity of the CC and C chemokines
suggests that they may play important roles in immunologic
responses.
Various CXC and CC chemokine genes, such as IFN--inducible
protein-10 (IP-10),3
monocyte chemotactic protein (MCP)-1, MCP-3, T cell activation 3,
RANTES, and macrophage-inhibitory protein (MIP) 1 have been
transduced into a variety of experimental tumors (13, 14, 15, 16, 17, 18).
Transfer of genes encoding antiangiogenic CXC chemokines (e.g., IP-10)
can block tumorigenicity in some cases (10, 11), whereas
results obtained with analogous CC chemokine gene transfers were
variable (15, 16, 17, 18). Nevertheless, these findings indicate
that the local secretion of some chemokines by engineered tumor cells
has the potential to affect tumorigenicity (15, 17) and
perhaps the induction of tumor-specific T cells (18). The
C chemokine lymphotactin has the ability to induce T (19)
or NK (20) cell migration in vitro and in vivo. Thus,
experimental expression of the gene for this chemokine by tumor cells
might be predicted to importantly affect the induction of
tumor-specific immune responses. In this study, we tested the effect on
tumor growth of transferring a lymphotactin gene expression cassette
into the highly tumorigenic mouse myeloma cell line SP2/0. Our results
indicate that this procedure induces specific protective responses that
are mediated by T cells, but also that lymphotactin receptor-expressing
neutrophils display some antitumor activities that are readily
discernable in T cell-deficient nude mice.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Rat monoclonal anti-mouse CD3 (145-2C11), CD4 (GK1.5), CD8 (3.155), NK (PK136), and neutrophil (RB6-8C5) Abs were purified by affinity chromatography from the ascites of hybridoma cell lines obtained from the American Type Culture Collection (ATCC, Manassas, VA) or Dr. R. Coffman (DNAX, Palo Alto, CA). The following reagents were purchased commercially: rat anti-mouse B7-1 (1G10), B7-2 (GL1), and ICAM-1 (3E2), and mouse anti-H-2Kd (SF1-1.1) and Iad (AMS-32.1) Abs, BD PharMingen (San Diego, CA); FITC- and peroxidase-conjugated goat anti-rat and anti-mouse IgG Abs, Bio/Can Scientific (Mississauga, Ontario, Canada); and recombinant mouse lymphotactin, R&D Systems (Minneapolis, MN). Myeloma cell line SP2/0 and B cell lymphoma cell line A20 (BALB/c mouse origin) were obtained from the ATCC and maintained in complete medium (DMEM-10% FCS-gentamicin, 50 µg/ml). Female BALB/c and athymic nude mice (4–6 wk old) were obtained from our institutional Animal Resource Center and Charles River Laboratories (St. Constant, Quebec, Canada), respectively. All mice were maintained in the animal facility at the Saskatoon Cancer Center (Saskatoon, Saskatchewan, Canada), and all experiments were conducted according to the guidelines of the Canadian Council for Animal Care.
Transfection of tumor cells with the expression vector pCI-Lptn
A 10-kb cDNA fragment encoding the full open reading frame of the mouse lymphotactin gene was obtained from Dr. A. Zlotnik (DNAX) and ligated into the vector pcDNA3.1 (Invitrogen, Carlsbad, CA) to form pcDNA3.1-Lptn. For transfection, 2 x 107 SP2/0 cells were resuspended in 0.7 ml PBS, mixed with 0.3 ml PBS containing 10 µg pcDNA3.1-Lptn or pcDNA3.1 DNA, and then electroporated at 250 V/125 µF capacitance (Gene pulser; Bio-Rad Laboratories, Richmond, CA). Two transfected cell lines, SP2/0-Lptn and SP2/0neo (control cell line), were obtained by selection for growth in complete medium containing 2.0 mg/ml G418, respectively.
Analysis of mRNA expression
Northern analysis of lymphotactin expression. Total cellular RNA was isolated from SP2/0-Lptn or control cells or tumors using a commercial kit (Qiagen, Mississauga, Ontario, Canada), denatured in formaldehyde, and electrophoresed on 1% agarose-formaldehyde gels. The integrity of the RNA samples in the gels was assessed under UV illumination; then the RNA was transferred to Nytran membranes (Schleicher & Schuell, Keene, NH). The EcoRI lymphotactin cDNA fragment was labeled with [32P]dCTP (Amersham, Arlington Heights, IL) by random priming and used to probe the blots. The membranes were hybridized overnight at 42°C in 6x SSC-5x Denhardt’s solution-0.5% SDS-20 µg/ml salmon sperm DNA. The filters were washed sequentially at 65°C in 3x SSC-0.1% SDS, 1x SSC-0.1% SDS, 0.3x SSC-0.1% SDS, and 0.1x SSC-0.1% SDS and then exposed to Kodak x-ray film until the desired exposures were obtained.
RNase protection analysis of chemokine expression. RNA was extracted from SP2/0 and SP2/0-Lptn tumors of nude mice as noted above and analyzed using a commercial kit (RiboQuant MultiProbe RNase Protection kit; BD PharMingen). Briefly, in vitro transcription of the RNAs for an array of chemokines (mCK5 MultiProbe Template Set; BD PharMingen) with [32P]UTP (Amersham) was conducted using with T7 RNA polymerase followed by phenol-chloroform extraction and ethanol precipitation. The probe mixture was adjusted to 3 x 105 cpm/µl and hybridized to the tissue RNA samples (6 x 105 cpm antisense RNA, 5 µg sample RNA); then the hybridized samples were digested with RNase followed by proteinase K treatment and phenol-chloroform extraction. After ethanol precipitation with 4 M ammonium acetate, the protected fragments were run on a 5.7% acrylamide-bisacrylamide urea gel, dried, and then visualized by autoradiography.
RT-PCR analysis of XCR1 expression.
Total RNA was obtained from nylon wool-purified T lymphocytes (>99%
purity as determined using the anti-CD3 Ab by FACS, data not shown)
and MACS-purified neutrophils. The neutrophils and RBC were first
separated from the peripheral monocytes with Ficoll-Paque gradient. The
neutrophils were then purified by lysing RBC with 0.84% ammonium
chloride to yield a neutrophil purity of 
95% and further purified
by MACS using Dynabeads M-450 (Dynal, Lake Success, NY) coated with the
antineutrophil Ab, to yield a final neutrophil purity of >99% as
determined using the antineutrophil Ab by FACS (data not shown).The
first-strand cDNA synthesis for the RT-PCR was performed with 5 µg
RNA using a commercial kit (Stratagene, La Jolla, CA), following the
manufacturer’s instructions. The PCR primers were specific for the
lymphotactin receptor gene XCR1 (sense primer, 5'-ctcct gtcta ctgcc
tgtgt tg-3'; antisense primer, 5'-tgact gttcg gtgtc tctgt ct-3'
21) and GAPDH (sense primer, 5'-caggt tgtct cctgc
gactt-3'; antisense primer, 5'-cttgc tcagt gtcct tgctg-3'). The PCR
conditions comprised 1 cycle at 94°C (5 min), 54°C (1 min), and
72°C (1 min) and 40 cycles at 94°C (1 min), 54°C (1 min), and
72°C (1 min). All PCR products were resolved on 1% agarose gels with
ethidium bromide staining.
Bioassays for lymphotactin
Cells. Splenic T lymphocytes were nylon wool purified from BALB/c mice as previously described (22) and resuspended in DMEM-0.1% BSA to 4 x 106 cells/ml. Mouse peripheral blood leukocytes were enriched by dextran sedimentation according to a standard protocol (23). Briefly, heparin-anticogulated mouse blood was mixed with equal volumes of 3% dextran T-500 (Pharmacia Biotech, Uppsala, Sweden) in 0.9% NaCl, and the RBC were allowed to sediment for 20 min at room temperature. The cells were then harvested from the leukocyte-rich plasma layer by centrifugation and washed and resuspended as above.
Chemotaxis assay. Chemotactic responses of T lymphocytes and neutrophils to lymphotactin or the tumor extracts were examined using modified Boyden microchemotaxis chambers (Neuroprobe, Gaithersburg, MD) and polyvinylpyrrolidone-free 5-µm pore size polycarbonate filters, essentially as described (24). Protein extracts of the SP2/0-Lptn and control SP2/0 tumors were prepared in 10 mM Tris-0.2 mM CaCl2 (pH 7.2) as previously described (25). The culture supernatants of SP2/0-Lptn and SP2/0 cells, protein extract samples (15 mg/ml), standards (recombinant lymphotactin; 1–1000 ng/ml), or positive control bacterial tripeptide FMLP (1 nM–10 µM) were diluted in DMEM-0.1% BSA and placed in the lower chambers of the wells, while the cells (T lymphocytes or neutrophils) were placed in the upper chambers. After incubation for 20 min (neutrophils) or 2 h (lymphocytes) at 37°C, the cells that had not migrated into the membranes were wiped from the upper surfaces; then the membranes were fixed and stained using a Diff-Quik kit (American Scientific Products, McGraw Hill, IL). For each sample, the numbers of cells associated with the membrane were enumerated by direct counting of at least nine x40 objective fields; the results are expressed as the mean number of cells/x40 field (±SEM). Essentially all (>99%) of the peripheral blood leukocytes that were adherent to the lower surfaces of the membranes in the 20-min assays were neutrophils, as determined by morphologic examination.
Animal studies
Tumorigenicity studies. In general, naive mice were inoculated s.c. on their right thighs with 1 x 106 SP2/0-Lptn, SP2/0neo, and SP2/0 tumor cells, respectively, and then monitored daily for tumor progression or regression. Two weeks later, the animals were sacrificed, and the tumors were carefully removed, weighed, and examined histologically. Tumor volumes (in cubic millimeters) were measured with a vernier caliper and calculated by the formula, tumor size = ab2/2, where a is the larger and b is the smaller of the two dimensions (26). The basic experiments were performed in BALB/c mice (n = 10), whereas other experiments involved T cell subset (CD4+ and CD8+) and NK-depleted BALB/c mice (2), nude mice, or neutrophil-depleted nude mice (each, n = 8). The T lymphocytes, NK cells, and neutrophils were depleted by injecting the mice i.p. on experimental days -1, 2, and 5 with 1 mg of the appropriate specific rat Abs (GK1.5, 3.155, PK136, and RB6-8C5), respectively; control mice were similarly injected with isotype-matched rat Abs.
To examine the protective immunity conferred by tumor inoculation, 1 x 106 SP2/0 or A20 tumor cells were inoculated into the right thighs of BALB/c mice (n = 8) that had previously rejected SP2/0-Lptn tumors. In some experiments, these SP2/0-Lptn tumor-immune mice were depleted of CD4+ or CD8+ T cells before SP2/0 tumor cell challenge. The tumors were removed and examined as described above. Each animal experiment was performed twice, and in each case the results from the independent experiments were consistent. Student’s t test was used to examine the statistical significance of the animal experiment results (2).
Histopathology and immunohistochemistry. To characterize the cellular infiltrates associated with SP2/0-Lptn tumor regression, 5 x 105 SP2/0-Lptn cells were injected into the kidney capsules (adjacent to the adrenal gland) of the mice. At different times thereafter, the kidneys/adrenal glands were removed and processed for histology or immunohistochemistry as previously described (27). For histopathology, the tissues were fixed in 10% neutral-buffered formalin, routinely processed to paraffin sections, and stained with hematoxylin-eosin. For immunohistochemical analysis, frozen sections were fixed with cold acetone, endogenous peroxidase activity was blocked by treatment with methanol-3% H2O2 (10 min), and the nonspecific staining was suppressed using 20% normal horse serum. Subsequently, the sections were incubated overnight at 4°C with either rat anti-mouse CD4 or CD8 Ab (10 µg/ml), then washed with PBS, and incubated with the biotinylated goat anti-rat IgG Ab for 30 min at room temperature. The sections were again washed and incubated in Vectastain avidin-biotin complex-peroxidase reagent (Vector Laboratories, Burlingame, CA) for 30 min; then the peroxidase activity was developed with freshly prepared 0.06% 3,3'-diaminobenzidine containing 0.1% hydrogen peroxide, and the sections counterstained with methyl green (0.1%). The sections were evaluated for the presence of brown diaminobenzidine precipitates indicative of CD4 and CD8 reactivity.
Immunologic assays
Phenotypic analysis of SP2/0-Lptn cells. We used FACS to quantitate the expression of MHC class I and II Ags, the costimulatory molecules B7-1 and B7-2, and ICAM-1 on SP2/0-Lptn tumor cells. To stain the cells, cells were first incubated for 1 h on ice with rat anti-B7-1, B7-2, or ICAM-1 or mouse anti-H-2Kd and Iad Abs (each, 2 µg/ml), then washed three times with PBS, and incubated for 1 h more on ice with FITC-conjugated anti-rat or anti-mouse IgG Ab (5 µg/ml). After another three washes with PBS, the cells were analyzed using an Epics XL FACS (Coulter, Burlington, Ontario, Canada).
Phenotyping the protective anti-SP2/0-Lptn response.
One week after inoculation of 1 x 106
SP2/0-Lptn cells in the footpads of the mice, lymphocytes were
harvested from their regional lymph nodes, washed, and resuspended in
complete medium. The lymphocytes were cocultured (in quadruplicate) in
96-well plates with 6000-rad-irradiated SP2/0 tumor cells (5 x
106 lymphocytes plus 2.5 x
106 SP2/0 cells/well). The culture supernatants
were harvested at 1 or 4 days and then pooled and assayed using
commercial ELISA kits for IFN- and IL-4 (Endogen, Woburn, MA),
respectively.
Cytotoxic T cell assay. Splenic lymphocytes were harvested from mice that had experienced SP2/0-Lptn tumor regression and from mice bearing SP2/0 tumors. The lymphocytes (5 x 106) were cocultured for 5 days with 6000-rad-irradiated SP2/0 cells (1 x 105) in 2-ml volumes of DMEM-10% FCS in 24-well plates (Costar, Cambridge, MA); then the T cells were harvested and used as effector cells in a chromium release assay. The target cells comprised 51Cr-labeled SP2/0 or A20 tumor cells, prepared by culturing the tumor cells for 1 h in the presence of 50 µl sodium [51Cr]chromate (36 mCi/ml; Amersham) and then washing them twice with DMEM. For the assay, 1 x 104 labeled target cells were incubated for 6 h with effector cells at various E:T ratios (triplicate cultures). The percentage specific lysis was calculated using the formula [(experimental cpm - spontaneous cpm)/(maximal cpm - spontaneous cpm)] x 100. The counts released spontaneously (i.e., in the absence of effector cells) were <10% of specific lysis; the maximal release was determined by adding 1% Triton X-100 to a set of wells.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Lymphotactin is a molecule known to chemoattract lymphocytes in
vivo (19). To test whether expression of this chemokine
within otherwise immunologically resistant tumors might render the
tumors susceptible to immune attack, we transfected SP2/0 myeloma cells
with a lymphotactin gene expression cassette and challenged BALB/c mice
with these transfected or nontransfected SP2/0 cells. We had determined
that the in vitro growth rate of the SP2/0-Lptn cells was similar to
the control SP2/0neo and the parental SP2/0 cells (doubling times,
12.4, 12.0, and 11.8 h, respectively) and had characterized the
expression of lymphotactin mRNA and product by the transfected cells in
vitro. The SP2/0-Lptn, but not the SP2/0 cells expressed high levels of
lymphotactin mRNA (Fig. 1
A).
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). To follow the
pathogenesis of the SP2/0-Lptn tumor response, we next injected these
cells into the kidney capsules of BALB/c mice and biopsied the
injection sites at various times thereafter. At 24 h
postchallenge, no leukocyte responses were evident at the SP2/0-Lptn
cell injection sites (Fig. 2B), but by 2 days
postchallenge some lymphocytes and neutrophils started to infiltrate
into the tumors (data not shown) and by 4 days postchallenge numerous
lymphocytes as well as neutrophils had infiltrated into the tumors
(Fig. 2C). Immunohistochemical analyses showed that these
lymphocytes comprised both CD4+ and
CD8+ cells (Fig. 3, A and B). However, no T cells were detected in a
4-day SP2/0 tumor growing in the kidney capsule of a control mouse
either by using anti-CD3 Ab immunostaining (Fig. 3C) or
by using anti-CD4 or CD8 Ab immunostaining (data not shown). Six
days after tumor cell injection, multiple areas of necrosis were seen
in the tumors (Fig. 2D), and by 9–10 day postchallenge the
tumor cells were no longer detectable, although numerous lymphocytes,
macrophages, and fibroblasts were present (Fig. 2E). No
cellular responses were observed in a 4-day SP2/0 tumor growing in the
kidney capsule of a control mouse (Fig. 2A). Taken together,
these data suggest that both CD4+ and
CD8+ T cells as well as neutrophils were involved
in the responses to the SP2/0-Lptn tumor cells in the BALB/c
mice.
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To assess the possible cellular mechanisms responsible for
SP2/0-Lptn tumor regression, we inoculated these cells into groups of
BALB/c mice that had been depleted of CD4+ or
CD8+ lymphocytes, NK cells and neutrophils,
respectively, and then tested their respective abilities to reject the
tumors. We found that there was no SP2/0-Lptn tumor growth in the
control Ab-treated, NK-depleted, and neutrophil-depleted mice, whereas
the SP2/0-Lptn cells essentially grew as aggressively as SP2/0 cells in
the CD4+- and
CD8+-lymphocyte-depleted naive mice (Table II). This indicates that at least the
initiation of immunologic rejection of SP2/0-Lptn tumor cells in naive
mice in the initiation phase of immune responses is dependent on both
CD4+ and CD8+ T cells. To
confirm the roles of the T cells in the tumor rejection response, we
repeated this SP2/0-Lptn challenge experiment in T cell-deficient nude
mice. As expected, the incidence of Sp2/0-Lptn, SP2/0neo, and SP2/0
tumor growth in these nude mice was equivalent (Table I
). However, we
found that there was a significant retardation of SP2/0-Lptn tumor
growth compared with the SP2/0 tumor growth in these nude mice (Fig. 4; p < 0.01). Histologic
data revealed that there were dense infiltrations of neutrophils in an
early stage (9 days) of SP2/0-Lptn tumors, and this was associated with
extensive tumor necrosis in a later stage (12 days) (Fig. 2, F and G). To determine whether the neutrophil
response was responsible for the retardation of SP2/0-Lptn tumor
growth, we next challenged neutrophil-depleted nude mice with these
cells and assessed the tumor growth. In these nude mice, there were no
neutrophil responses to the tumor cells (Fig. 2H), and
SP2/0-Lptn tumors grew as aggressively as SP2/0 tumors (Fig. 4). Taken
together, these data confirm that T cells are important to SP2/0-Lptn
tumor cell rejection but also clearly demonstrate that neutrophils play
a role in this response.
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Lymphotactin has not been reported previously to affect
neutrophils, although it is well known for its effects on lymphocytes
(19). Our data confirmed that both recombinant
lymphotactin and SP2/0-Lptn cell culture supernatants were able to
chemoattract T cells in a dose-dependent manner. The amount of
lymphotactin in SP2/0-Lptn cell culture supernatants was estimated to
be 500 pg/106 cells during 24 h in
culture (Fig. 5A). To validate
this apparent lymphotactin-mediated neutrophil response, we first
confirmed using RT-PCR that highly purified mouse neutrophils (>99%
purity) expressed substantial levels of the lymphotactin receptor XCR1
as did the mouse T lymphocytes (Fig. 1C). We then assessed
the neutrophil chemotactic properties of recombinant lymphotactin, as
well that of aqueous extracts of SP2/0-Lptn tumors resected from nude
mice. Our data confirm that both recombinant lymphotactin and
SP2/0-Lptn tumor extracts (but not SP2/0 tumor extracts) were able to
chemoattract peripheral blood neutrophils from BALB/c mice in a
dose-dependent fashion as did the chemotactic bacterial tripeptide FMLP
(Fig. 5, B–D). To determine whether other high
profile neutrophil chemokines were expressed within the SP2/0-Lptn
tumors grown in nude mice, we probed the RNA extracted from the tumors
with a panel of chemokine probes (MIP-1, MIP-2, IP-10) using an
RNase protection assay. There was no detectable expression within the
SP2/0-Lptn tumors of MIP-1, MIP-2, or IP-10 (i.e., neutrophil
chemokines (13, 28, 29)), whereas there was significant
expression of mRNA for lymphotactin.
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To gain insights into the immunologic basis of SP2/0-Lptn tumor
regression, we assessed a number of parameters associated with this
tumor regression. First, we used FACS to examine the expression by
SP2/0 and SP2/0-Lptn cells of MHC class I and II Ags, the costimulatory
molecules B7-1 and B7-2, and the adhesion molecule ICAM-1 (i.e.,
molecules important to immunogenic processes). Both SP2/0 and
SP2/0-Lptn cells expressed MHC class I Ags, but MHC class II, B7-1,
B7-2, or ICAM-1 molecules were not detectable in either population, and
the SP2/0-Lptn cells expressed slightly but not remarkably enhanced MHC
class I relative to the SP2/0 cells (data not shown). This indicates
that the loss of tumorigenicity of SP2/0-Lptn tumor cells was not due
to an alteration of immunogenicity. We next examined the phenotype of
the SP2/0-Lptn cell-specific responses in SP2/0-Lptn-immune mice by
assessing the IFN-/IL-4 cytokine profiles released by regional
lymphocytes of tumor-exposed animals. The SP2/0-Lptn-responsive T cells
in the animals undergoing SP2/0-Lptn tumor regression secreted high
levels of IFN-, but little IL-4 (1120 ± 130 pg/ml IFN-,
<33 pg/ml IL-4), whereas analogous cells from naive (data not shown)
or SP2/0 tumor-bearing mice secreted little IFN- (110 ± 45
pg/ml IFN-, <33 pg/ml IL-4) (Table III), suggesting that the
anti-SP2/0-Lptn immune response was of a Th1 type.
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). These results clearly demonstrate a SP2/0
tumor-specific protective immunity in BALB/c mice previously vaccinated
with SP2/0-Lptn tumor cells.
To gain insights into the immunologic basis of SP2/0-Lptn-inducible
protection, we assessed a number of parameters associated with the
induction of such immune responses. First, we used anti-CD4,
anti-CD8, or irrelevant control Abs to deplete SP2/0-Lptn-immune
mice of their respective lymphocyte subpopulations and then challenged
the mice with SP2/0 cells. The SP2/0-Lptn tumors did not grow in the
control Ab-treated (tumor incidence, 0 of 8) or anti-CD4-Ab-treated
(incidence, 0 of 8) groups (Table II), but significant
tumor growth was clearly present in the CD8+
lymphocyte-depleted mice (tumor incidence, 8 of 8). Furthermore,
following coculture for 5 days with irradiated SP2/0 cells, splenocytes
from the SP2/0-Lptn-immune mice displayed highly significant cytotoxic
activity against SP2/0 cells (78% specific killing; E:T ratio, 100)
but not against irrelevant control A20 cells. Lymphocytes from mice
bearing SP2/0 cell tumors showed only low SP2/0-specific killing
(10%), and splenocytes from naive mice did not show any SP2/0-specific
responses (data not shown). Thus, these data indicate that
anti-SP2/0 tumor protection in the SP2/0-Lptn-vaccinated mice was
largely mediated by SP2/0 tumor-specific CTLs.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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secretion. Although lymphotactin is chemotactic
for T cells, recently it was reported that lymphotactin can also
differentially regulate cytokine secretion by
CD4+ and CD8+ T cells
(30). It dampens the IL-2 and IFN- responses of
CD4+ T cells, although IFN- expression in the
lymphotactin-stimulated CD4+ T cells still
outstrips IL-4 release by 5-fold but also augments the IL-2 responses
of CD8+ T cells (the IFN- responses of
CD8+ T cells were not examined). It is feasible
then that the IFN- expression observed in our study was attributable
to both Th1 and Tc1 cells that were activated via the SP2/0-Lptn
immunization. The CD4+ cells were critical for
the induction of the tumor-specific immunity, because their depletion
before initial exposure to the SP2/0-Lptn cells also depleted the
ability to reject the tumor cells. The effector phase of the tumor
rejection was mediated by CD8+ cells and not at
all affected by CD4+ cell depletion. Previous
studies on tumor resistance following chemokine gene transfer neither
defined the role of T cells nor analyzed the type 1 or type 2 nature of
these responses (31, 32, 33, 34). Collectively, our results
demonstrate that the C chemokine lymphotactin has natural adjuvant
activities that result in augmented antitumor immune responses.
Neutrophil infiltration into tumors has been frequently reported in
studies with tumor cells engineered to secrete cytokines and
chemokines. Thus, the potential involvement of neutrophils in antitumor
immune responses has begun to draw more attention. This neutrophil
response has been documented in the context of tumor cells engineered
to secrete IL-2, IL-4, IL-8, GM-CSF, MIP-1, or MCP-3, wherein the
engineered cells showed reduced tumorigenicity or underwent tumor
regression in syngeneic mice (1, 17, 32, 33, 34, 35).
Nevertheless, neither the mechanism(s) of neutrophil recruitment nor
the potential role(s) of these cells in antitumor responses were
elucidated in these studies. Recently, Shinohara et al. demonstrated
that expression of MIP-1 and MIP-2 was up-regulated in neutrophils
that infiltrated GM-CSF-transfected tumors and that the observed
neutrophil response preceded a subsequent macrophage effector response.
Their data indicated that the neutrophil chemokines in fact mediated
the chemoattraction of the macrophages into the tumors
(21), which illustrates one potential role that
neutrophils can play in antitumor responses. More recently, Lee et al.
(36) demonstrated that neutrophil infiltration occurred at
injection sites of IL-8 transgene-expressing human ovarian cancer cells
in nude mice and that this was associated with significantly reduced
tumorigenicity in vivo. Our data clearly demonstrate that neutrophils
express significant levels of the lymphotactin receptor XCR1, and this
chemokine is chemotactic for not only T cells but also neutrophils,
both in vitro and in vivo. Furthermore, we have documented that
antineutrophil Abs can block the retardation of SP2/0-Lptn tumor growth
observed in nude mice. Our study thus provides direct evidence that
cooperates earlier suggestions that neutrophils may play some role in
inhibition of tumor growth in mice. Although neutrophils recruited as a
consequence of lymphotactin gene transfer exert some antitumor
activity, they do not play a requisite role in tumor regression in
fully immunocompetent BALB/c mice, because in these animals
CD4+CD8+ T cells are
themselves able to accomplish this function. We did not address the
mechanisms by which the neutrophils effected their antitumor activities
in nude mice, but they may involve the release of granules, oxygen free
radicals, or proteases, each of which could be pathogenic for tumor
cells (37).
Recent clinical studies showed that CD8+ T cell
infiltration of tumors contributed to better survival of patients
(38). In systemic adoptive immunotherapy, it has generally
been believed that the antitumor efficacy of the transferred T cells
is, to a large extent, determined by their ability to extravasate into
the tissues where tumors reside. Clinically, the therapeutic efficacy
of this approach is still very limited (objective response rate of
30%; Ref. 39), most probably because the proportion of
transferred T cells that accumulate within the tumors is rather small
(40, 41). Because an efficient infiltration of tumors by
systemically transferred tumor-reactive T lymphocytes is required for
antitumor efficacy (42), the local secretion of
lymphotactin at tumor sites (as could be effected, for example, by
adenovirus-mediated lymphotactin gene transfer (43) could
perhaps increase the infiltration into tumors of adoptively transferred
tumor-specific T cells and thus improve the efficiency of adoptive
cellular immunotherapies. We have already shown, for example, that
combined immunotherapies using both a cancer vaccine and gene transfer
can be effective in enhancing responses against established tumors
(44). In that study, we injected an adenovirus expressing
TNF- intratumorally and thereby enhanced T cell infiltration into
tumor masses (44). It remains to be determined whether a
similar approach using lymphotactin gene transfer would also induce
effective responses against established tumors, but the roles in
immunologic responses demonstrated herein and by others for this
chemokine suggest that this may well be a fruitful avenue of
investigation.
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Acknowledgments |
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Footnotes |
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2 Address correspondence and reprint requests to Dr. Jim Xiang, Saskatoon Cancer Center, 20 Campus Drive, Saskatoon, Saskatchewan S7N 4H4, Canada. E-mail address: JXiang{at}SCF.SK.CA
3 Abbreviations used in this paper: IP-10, IFN--inducible protein-10; MIP, macrophage-inhibitory protein; MCP, monocyte chemotactic protein.
Received for publication November 8, 2000. Accepted for publication April 27, 2001.
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) or SP2/0 (
) tumor cells and
followed the growth of the tumors with time. Another group of eight
neutrophil-depleted nude mice were similarly injected with SP2/0-Lptn
tumor cells (•). Tumor size was measured as described in
Materials and Methods.
) and SP2/0 tumors (
)
grown in nude mice (D). Aqueous extracts of SP2/0 tumors
did not contain detectable neutrophil chemoattractant activities.
E, The anti-lymphotactin Ab blocks the chemotactic effect of
SP2/0-Lptn tumor extracts on neutrophils.