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*
Department of Clinical and Biological Sciences, University of Torino, Orbassano, Italy;
Departments of Cell Biology and Molecular Biology, Human Genome Sciences, Inc., Rockville, MD 20850;
Department of Oncology and Neurosciences, University of Chieti, Chieti, Italy; and
§
Institute for Cancer Research, University of Bologna, Bologna, Italy
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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and IL-2 when restimulated by
TSA-pc. The ability of LEC to markedly improve recognition of poorly
immunogenic cells by promoting APC-T cell cross-talk suggests that it
could be an effective component of antitumor
vaccines. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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and ß chemokines) have been extensively
characterized. ß chemokines constitute the larger family. They
display two contiguous cysteines (C-C motif) and most of them are coded
by genes clustered on human chromosome 17q11-2 (2). The
gene encoding the LEC ß chemokine (also known as NCC-4, HCC-4, and
LMC) has been located in this cluster (2), and its genomic
organization (3) and amino acid sequence (4)
have been determined. LEC is chemotactic for human monocytes and
dendritic cells and not neutrophils (5, 6, 7). This suggests
that LEC may be involved in inflammation. Paradoxically and by contrast
with other chemokines, the expression of LEC mRNA is up-regulated by
IL-10 (5). Comparison of the effects of chemokines by transducing a variety of tumor cells with their genes has shown that their local release influences tumor growth, angiogenesis, and immunogenicity. The effect achieved is highly dependent on the type of chemokine (1). The present study was designed to determine whether local secretion and accumulation of LEC influences the growth and immunogenicity of TSA, an aggressive mouse adenocarcinoma line, whose cells have been extensively engineered to release many other cyto/chemokines and whose behavior and immunogenicity have been carefully compared (8, 9, 10).
We show that the release of LEC by engineered TSA cells quickly induces their rejection by syngeneic mice and inhibits their metastatic spread. Rejection is associated with an impressive infiltrate of macrophages, dendritic cells, T cells, and polymorphonuclear (PMN)4 leukocytes, and is attributable to the last two populations. It is followed by the establishment of an effective and specific immune memory against TSA wild-type parental cells (TSA-pc). The unique effectiveness of this rejection and induction of a specific immune memory suggest that LEC could be made an effective component of gene-engineered antitumor vaccines.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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TSA-pc are an aggressive and poorly immunogenic cell line established from a moderately differentiated mammary adenocarcinoma that arose spontaneously in a multiparous BALB/c mouse (10). They express MHC class I, but not class II molecules, secrete G-CSF and GM-CSF, TGF-ß (11), basic fibroblast growth factor and vascular endothelial growth factor (12), but not LEC (data not shown) and do not stimulate a syngeneic antitumor response in vivo, nor in mixed lymphocyte-tumor cell cultures (11). F1-F is a newborn BALB/c mouse-derived skin fibroblast cell line that spontaneously transformed after the 15th in vitro passage. Its cells do not immunologically cross-react with TSA-pc and they were therefore used as controls (13). Confluent monolayers of TSA or F1-F cells treated with a 0.25% solution of trypsin in HBSS (Sigma, Milan, Italy) were used for in vitro and in vivo experiments. Their minimal 100% tumor-inducing doses in BALB/c mice are 5 x 104 and 1 x 104 cells, respectively.
Transfection of TSA-pc cells
The LEC ß chemokine gene used in this study was cloned by Human Genome Sciences (Rockville, MD) through an expressed sequence tags database search by a high degree of homology to the ß chemokine genes. To generate the LEC expression vector, the LEC open reading frame was amplified as a HindIII/XbaI fragment and subcloned into likewise digested pcDNA3. Insert sequences were confirmed by double-stranded sequencing. pcDNA3 expression vectors contain the neomycin resistance gene and were used for TSA-pc transfection. Cells were plated at a density of 6 x 105 cells/100-mm tissue culture plate and incubated overnight at 37°C in DMEM with 4.5 g/l glucose and 10% FBS (BioWhittaker, Walkersville, MD). They were then suspended in OptiMEM without FBS and transfected using LipofectAMINE reagent (Life Technologies, Rockville, MD). Forty-eight hours later, they were trypsinized (Sigma), split 1:10, and plated in DMEM with 1 mg/ml of geneticin (G418; Life Technologies). Clones were isolated and subcloned by limiting dilution 15–20 days later. TSA-neo is a control clone transfected with the neomycin resistance gene only.
Clone selection
The amount of LEC protein secreted in 1 ml medium by 1 x 105 seeded transfected TSA cells after 48 h of culture was evaluated by ELISA using polyclonal rabbit Ab against LEC, and recombinant LEC proteins (all from PeproTech, Rocky Hill, NJ). One representative transfected clone denominated TSA-LEC was selected for use in the experiments, since it consistently releases 60–70 ng of LEC as determined by repeated determinations during the experimental period (data not shown).
Mice
Seven-week-old female nu/nu (CD1) and BALB/cAnCr (H-2d) mice (Charles River Breeding Laboratories, Calco, Italy) were treated in accordance with European Union guidelines. When required, starting 2 days before tumor challenge, 1 h before and then at weekly intervals, a few mice received i.p. injections of 100 µg anti-CD8 (TIB-105 hybridoma, Lyt 2; American Type Culture Collection, Manassas, VA), anti-CD4 (GK1.5 hybridoma, L3T4; American Type Culture Collection), anti-PMN mAb (RB6–8C5 hybridoma, kindly provided by R. L. Coffman, Dnax, Palo Alto, CA), or ascitic fluid eluting from anionic exchange columns (DE 52; Whatman, Maidstone, U.K.). Flow cytometry of their residual blood and spleen cells showed that target leukocytes were selectively decreased to 1/5000 of peripheral blood leukocytes during treatment. Immunosuppressed mice were fed with sterilized food pellets and tap water ad libitum.
In vivo evaluation of tumor growth and metastases
Mice were challenged s.c. in the left flank (primary challenge) or right flank (secondary challenge) with 0.2 ml of a single-cell suspension containing the indicated number of tumor cells. The cages were coded, and the incidence and the growth of tumors were evaluated twice weekly in a blind fashion. Neoplastic masses were measured with calipers in the two perpendicular diameters for 120 days. At the end of this period, tumor-free mice were classed as survivors. Latency and survival times were considered as the periods (in days) between challenge and the growth of neoplastic masses of 3- and 10-mm mean diameter, respectively. Only mice that eventually developed tumors were considered. Mice were killed for humane reasons when the tumor exceeded 10-mm mean diameter. Metastases were induced by injection of 5 x 104 TSA cells in a lateral tail vein. Lung nodules were evaluated 21 days later on dissected lung lobes contrasted with black india ink under a stereoscopic microscope.
Morphological analysis
For histologic evaluation, tissue samples were fixed in 10%
neutral buffered Formalin, embedded in paraffin, sectioned at 4 µm,
and stained with hematoxylin and eosin or Giemsa. For
immunohistochemistry, acetone-fixed cryostat sections were incubated
for 30 min with anti-CD4, anti-CD8a (Sera-Lab, Crawley Down,
Sussex, U.K.), anti-Mac-1 (anti-CD11b/CD18), anti-Mac-3,
anti-Ia (Boehringer Mannheim, Milan, Italy),
anti-polymorphonuclear leukocyte (RB6-8C5), anti-endothelial
cell (mEC-13.324, provided by Dr. A. Vecchi, Negri Nord, Italy),
anti-dendritic cells (NLDC 145; Cederlane Laboratories, Ontario,
Canada), anti-IL-1ß (Genzyme, Cambridge, MA), anti-TNF-
(Immuno Kontact, Frankfurt, Germany), anti-IFN- (provided by Dr.
S. Landolfo, Torino University, Italy), anti-IL-6 (PharMingen,
Milan, Italy), and anti-iNOS (Transduction Laboratories, Lexington,
KY) Abs. After washing, the cryostat sections were overlaid with
biotinylated goat anti-rat, anti-hamster, and anti-rabbit
or horse anti-goat Igs (Vector Laboratories, Burlingame, CA) for 30
min. Unbound Ig was removed by washing, and the slides were incubated
with avidin-biotin complex/alkaline phosphatase (Dako, Glostrup,
Denmark). Quantitative studies of the immunohistochemically stained
sections were performed by three pathologists in a blind fashion on
three or more samples from distinct mice by evaluating 10 randomly
chosen fields in each sample. Individual cells were counted under a
microscope (x40 objective and x10 eyepiece; 0.18 mm 
per
field). The expression of cytokines and adhesion molecules was classed
as absent (-) and scarcely (+), moderately (++), and frequently (+++)
present on cryostat sections tested with the corresponding Ab.
Cellular cytotoxicity
Effector lymphocytes were generated by culturing 1 x 107 responder splenic (Spc) and lymph node cells with 5 x 105 mitomycin C (Mit-C)-treated stimulator TSA-pc cells for 6 days. Cytotoxic activity was assayed in a 48 h [3H]TdR release assay (11) by mixing effector lymphocytes with 5 x 103 labeled target cells at 50:1, 25:1, 12:1, and 6:1 E:T ratios in round-bottom 96-well microtiter plates in triplicate. Specific lysis was determined as previously described in detail, and the values were expressed as LU20/107 cells calculated as previously described (11).
Cytokine production
In one set of experiments, 1 x 106
Spc from normal mice and mice that had rejected TSA-LEC injected 30
days earlier were cultured in 24-well plates (Falcon; Becton Dickinson,
Meylan, France) for 48 h either in 1 ml of medium or with 2.5
x 104 Mit-C stimulator TSA-pc. The amount of
IL-2, IFN-, IL-4, and IL-10 released was evaluated with
cytokine-specific ELISA kits (PharMingen).
Immune sera
Mice were injected in the left flank with a single-cell suspension of 1 x 105 trypan blue dye-excluding, proliferating TSA-LEC, or Mit-C TSA-pc in 0.2 ml HBSS. Seven and 30 days after the challenge, sera were collected from groups of five mice and pooled. Normal sera were pooled from five age-matched, untreated mice. The specific TSA-pc-binding potential (sbp) of the sera was evaluated by flow cytometry after indirect immunofluorescence as described previously (11). Briefly, 2 x 105 cultured TSA-pc were washed twice with cold HBSS supplemented with 2% BSA and 0.05% sodium azide and stained with 50 µl of 1:10 dilution in HBSS-azide-BSA of normal or immune sera. FITC-F(ab') (2) rabbit anti-mouse Ig (Dako) was used as second-step Ab. Labeling steps were followed by incubation for 30 min at 4°C and two washes with cold HBSS-azide-BSA. Stained cells were analyzed with a FACScan flow cytometer (Becton Dickinson, Mountain View, CA) and the sbp/50 µl serum was calculated as follows: [(%positive cells with test serum) (fluorescence mean)] -[(% positive cells with normal serum) (fluorescence mean)] x serum dilution. Dead cells were gated on the basis of propidium iodide labeling. A total of 1 x 104 viable cells was analyzed in each evaluation. Each titration was performed four times, and the results are expressed as mean sbp x 10-3/ml ± SEM.
Statistical analysis
The significance of differences in tumor takes was evaluated by
Pearson’s 
2 test, those in survival and
latency time, in vitro data and those in the number of
tumor-infiltrating cells by a two-sample Student’s t test,
and those in the number of experimental metastases by Wilcoxon’s
test.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The in vitro doubling times, assessed as previously reported (14), of TSA-LEC cells were similar to those of TSA-neo and TSA-pc, suggesting that LEC secretion has no major direct effect on TSA cell proliferation (data not shown). Flow cytometry revealed a small increase of MHC class I on TSA-LEC cell membranes as compared with TSA-pc (data not shown). The in vitro and in vivo data obtained with TSA-pc and TSA-neo never differed significantly. For the sake of simplicity, only those obtained with TSA-pc are shown.
To evaluate whether LEC secretion affects the ability of TSA cells to
give rise to a tumor, syngeneic BALB/c mice were challenged s.c. with a
TSA-pc dose two times higher than the minimal 100% tumor-inducing
dose. All of these mice developed tumors that increased in size with a
similar progression pattern, whereas only 20% of mice challenged with
TSA-LEC cells displayed tumors (Table I
).
These few tumors were characterized by delayed growth patterns and a
latency time almost double that of TSA-pc.
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Variously immunosuppressed mice were challenged with TSA-LEC
cells to characterize the leukocyte subpopulations mainly involved in
their rejection. T-deficient nu/nu mice challenged with
TSA-LEC cells developed tumors that developed progressively but more
slowly than those challenged with TSA-pc (Table I
). TSA-LEC rejection
by BALB/c mice was not impaired by CD4+ and
asialo GM1+ depletions, whereas it was abolished
by CD8+ depletion. Rejection does not simply rest
on CD8+ cells, however, since it was also
significantly impaired by PMN depletion (Table I).
Morphologic features associated with TSA-LEC cells growth and rejection
TSA-pc grow and kill BALB/c mice. Three days after challenge,
TSA-pc cells have already formed solid tumors with numerous mitotic
figures. At day 7, the tumor invaded subcutaneous fibroadipose tissue
and epidermis (Fig. 1a). This
growth was sustained by a well-developed and evenly distributed
vascular network. Macrophages and a few granulocytes and lymphocytes
infiltrated the tumor mass peripherally, where endothelial microvessels
faintly stained for the ICAM-1 adhesion molecule (Table III).
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b). A marked
infiltration of dendritic cells was detected, particularly within the
few surviving aggregates of tumor cells (Fig. 1d).
CD8+ and CD4+ lymphocytes,
macrophages, and granulocytes were widely recruited at the TSA-LEC
tumor site (Table III and Fig. 1f) and penetrated to the
center of the tumor. Lymphocytes were massively present and frequently
tightly close to TSA-LEC cells. This impressive leukocyte recruitment
was associated with an intense proinflammatory cytokine production and
macrophagic inducible NO synthase (iNOS) activation (Table III).
Inducible endothelial adhesion molecules, namely, endothelial leukocyte
adhesion molecule-1, VCAM-1 and ICAM-1, were intensely expressed on
most of the TSA-LEC tumor vessels (Table III and Fig. 1h).
Microvessel density was not appreciably different from that observed
for TSA-pc tumors.
In nu/nu mice, TSA-LEC cells grow progressively.
Infiltrating macrophages and granulocytes were remarkably less numerous
than in BALB/c mice. A weak production of proinflammatory cytokines was
detected in the tumor microenvironment, where only iNOS was strongly
expressed. No adhesion molecules were found on tumor microvessels
(Table III).
Acquisition of an immune memory to TSA-pc
At progressive times after TSA-LEC challenge in the left flank,
mice were rechallenged with TSA-pc in the right flank to evaluate the
potential of the systemic reaction elicited by TSA-LEC. Tumor takes of
TSA-pc were only evaluated in mice in which TSA-LEC cells did not grow
in the left flank. No protection was found when TSA-pc were injected
simultaneously with TSA-LEC cells or 1 day after, whereas 75% of mice
rejected TSA-pc when challenged 6 days later. All mice were protected
against a challenge performed after 10 or 30 days. A specificity check
with the unrelated F1-F tumor cell challenge displayed the selectivity
of this immune memory (Table IV).
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Mice that had rejected a primary TSA-LEC challenge in the left
flank received a secondary TSA-pc challenge in the right flank 30 days
later. At 72 h, the TSA-pc growth area was heavily infiltrated by
reactive cells and a few aggregates of tumor cells with marked signs of
injury were evident. The number of lymphocytes, and particularly that
of CD8+ lymphocytes, was significantly higher
than that found after primary TSA-pc challenge in nonimmunized mice.
Endothelial adhesion molecules along with proinflammatory cytokines,
especially IFN-, were strongly expressed (Table III). When mice that
had rejected TSA-LEC cells 30 days before were selectively
immunosuppressed during the secondary challenge, it was found that
memory TSA-pc rejection was not affected by NK cell depletion, whereas
it was impaired by CD4+ and
CD8+ lymphocyte and PMN cell depletion
(Table IV).
Both fresh and TSA-pc-restimulated Spc from mice that had rejected
TSA-LEC cells displayed a marked and specific cytolytic activity
against TSA-pc (Table V). Following
restimulation with TSA-pc, these Spc released 5 times more IL-2 and 10
times more IFN- than restimulated Spc from normal mice (Fig. 2). Neither IL-4 nor IL-10 were released
(data not shown).
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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In immunocompetent mice, TSA-LEC cells display an impressive ability to
promote monocyte, dendritic cell, T cell, and granulocyte influx into
the tumor site. These findings do not contradict the in vitro data on
the chemotactic activity of LEC (5, 6), but certainly seem
more remarkable than might have been expected. This consideration
coupled with the marked differences in infiltrating cells observed in
nu/nu and immunocompetent mice points to a major role for T
cells in orchestrating the magnitude of the infiltrate and the efficacy
of the reaction. In immunocompetent mice, TSA-LEC cells elicit a marked
local production of IL-1ß, TNF-, and IFN- and induce the
expression of adhesion molecules on tumor vessels. Leukocyte adhesion,
transmigration, and recruitment appear to be guided by these two key
events. In contrast, up-modulated expression of adhesion molecules on
tumor vessels, recruitment of macrophages and granulocytes, and
cytokine production are all almost negligible in T cell-deficient
nu/nu mice.
Both in vitro (5, 7) and in vivo, LEC does not appear to be a typical chemotactic agent as it is endowed with a few peculiar features: 1) its in vitro chemotactic activity on macrophages and dendritic cells takes place at abnormally high concentrations when compared with other chemokines; 2) its mRNA is constitutively expressed by a wide range of tissues; and 3) in contrast to the down-regulatory effect of IL-10 on the expression of most chemokines, LEC expression is up-regulated by IL-10 (5, 7).
The sustained accumulation of LEC in the TSA-LEC tumor microenvironment
may account for the recruitment of reactive cells leading to tumor
rejection. Yet, how are T lymphocytes so impressively recruited despite
the evidence that LEC is only slightly or occasionally chemotactic for
resting T cells in vitro (5, 6, 7)? High local concentrations
of LEC could induce downstream mediators, leading to T cell
recruitment. Macrophages recruited and activated by LEC may produce
proinflammatory factors, leading to dendritic cell maturation
(15). By releasing DC-CK1 and other chemokines, dendritic
cells may subsequently attract and specifically activate T lymphocytes
in the tumor microenvironment (16, 17, 18, 19). This possibility
is endorsed by immunohistochemical data showing that TSA-LEC cells also
recruit macrophages and activate TNF-, IL-1ß, and iNOS production
in T lymphocyte-deficient nu/nu mice. Alternatively,
macrophages and dendritic cells attracted by LEC may migrate to
lymphoid organs and present TSA Ag peptides to T cells
(20). These activated T cells then home into the tumor
area, release proinflammatory cytokines, and orchestrate tumor
destruction.
The well-organized capillary network of a TSA-LEC tumor suggests that
angiogenesis inhibition is not the means by which LEC induces TSA
regression, unlike many other cytokines such as IFN-, IFN-
(21), and IL-12 (9). Data from selectively
depleted syngeneic mice point to a major role of the
CD8+ subset and underscore the significant role
of PMN cells in TSA-LEC rejection. This finding offers further evidence
of the importance of the PMN-T cell cross-talk in cytokine- and
chemokine-elicited tumor rejection (8, 21).
TSA-pc are poorly immunogenic in immunization-protection tests and
unable to stimulate proliferation and lymphokine release by syngeneic
lymphocytes (10). The early onset of a strong and systemic
memory to TSA-pc following TSA-LEC challenge suggests that LEC
accelerates events that underlie a cross-talk between T cells and APCs
(22, 23). Morphologic observations of TSA-pc memory
rejection showed the absence of the macrophage and dendritic cell
infiltrate that was the hallmark of the primary TSA-LEC rejection,
whereas a conspicuous CD8+ and
CD4+ lymphocyte infiltrate and a distinct
expression of proinflammatory cytokines were evident. The importance of
CD8+ T cells in secondary TSA-pc rejection, along
with the strong lytic activity of Spc associated with their ability to
release IFN- and IL-2 but not IL-4, suggests an immune memory
deflected toward a Th1-type reaction. However, depletion experiments
also underscore a crucial role of CD4+
lymphocytes and PMN cells. This suggests that TSA-LEC triggers a more
articulated memory mechanism. Indirect presentation of tumor Ags may
trigger Th cells to release IFN- and other cytokines through which
the antitumor activity helper T cells attract, activate, and guide PMN
cells (13, 21, 24, 25, 26). Finally, the titer of anti-TSA
Ab quickly elicited by TSA-LEC is markedly higher than that observed
after challenge with TSA cells engineered to release cytokines
eliciting a Th1 or Th2 response, such as IL-4 (13), IL-10
(27), and IL-12 (9). These Abs could provide
further guidance for PMN-dependent tumor rejection
(13).
The rejection of tumor cells engineered to release cytokines and
chemokines is often followed by establishment of a systemic immune
memory specific to a subsequent challenge by wild-type tumor cells. The
features of this memory are dictated by the factor released and the
mechanisms of tumor cell death (8, 28). Since TSA-pc have
been engineered to release many factors (10), their
immunogenic potential can be compared (8, 9, 29). The
efficacy of the memory elicited by TSA-LEC cells appears to be superior
to that elicited by TSA cells engineered to release other powerful
memory-inducing cytokines, such as IL-2, IFN-, and IL-12
(8, 9, 27). This makes LEC a candidate for use as a very
effective component of engineered tumor vaccines.
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Acknowledgments |
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Footnotes |
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2 Address correspondence and reprint requests to Dr. Mirella Giovarelli, Department of Clinical and Biological Sciences, University of Torino, Ospedale San Luigi Gonzaga, 10043, Orbassano, Italy. E-mail address:
3 Current address: Ares-Serono International SA, 12 Chemin des Aulx, CH-1228 Plan-les-Ouates, Geneva, Switzerland.
4 Abbreviations used in this paper: PMN, polymorphonuclear; iNOS, inducible NO synthase; Mit-C, mitomycin C; Spc, splenic cells; sbp, specific-binding potential; TSA-pc, TSA parental cells.
Received for publication September 23, 1999. Accepted for publication December 30, 1999.
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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. Proc. Natl. Acad. Sci. USA 96:8633.-interferon gene or admixed with conventional adjuvants. Cancer Res. 54:6022.This article has been cited by other articles:
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