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Journal of Cancer Therapy, 2012, 3, 337-342
http://dx.doi.org/10.4236/jct.2012.324044 Published Online September 2012 (http://www.SciRP.org/journal/jct)
337
Low Dose of IL-12 Stimulates T Cell Response in Cultures
of PBMCs Derived from Non Small Cell Lung Cancer
Patients*
Lucia D’Amico1, Enrico Ruffini2, Riccardo Ferracini3, Ilaria Roato1#
1CeRMS (Center for Research and Medical Studies), A.O. Città della Salute e della Scienza di Torino, Torino, Italy; 2Department of
Toracic Surgery, A.O. Città della Salute e della Scienza di Torino, Torino, Italy; 3Department of Orthopaedics, A.O. Città della Sa-
lute e della Scienza di Torino, Torino, Italy.
Email: #roato78@libero.it
Received July 31st, 2012; revised August 30th, 2012; accepted September 10th, 2012
ABSTRACT
Cancer induces tolerance by suppressing immune function, modulating the T helper activity and causing an imbalance
of cytokines produced by T cells. IL-12 is an immune regulatory cytokine with potent anti-tumor activity and its signal-
ling network leads to polarization of naïve CD4 T cells into Th1. In pre-clinical studies, administration of recombinant
IL-12 by intravenous injection or IL-12 plasmid DNA by intra-tumoral injection showed some anti-tumor effects,
measurable immunological responses, but also important dose-dependent side effects. We investigated the ability of low
doses of IL-12 to modulate the T cell subpopulations in cultures of PBMCs derived from Non Small Lung Cancer
(NSCLC) patients and to induce lysis of lung adenocarcinoma cells by T cells. PBMCs were stimulated with different
doses of IL-12 and T cell phenotype was evaluated. IL-12 at 0.01 pg/ml significantly increased the number of CD4 and
CD8 T cells, in particular of CD4/IFN
producing cells. IL-12 did not stimulate T regulatory, but it increased the lysis
of lung adenocarcinoma cells induced by T cells. Our results showed that low doses of IL-12 modulated T cell sub-
populations in vitro and it increased their lytic activity on adenocarcinoma cells. Thus we hypothesize the use of low
dose of IL-12 as a therapeutic tool against pathologies characterized by a T cell imbalance, in order to promote an im-
muno-modulation.
Keywords: Interleukin-12; Immuno-Modulation; T Helper; T Regulatory; Anti-Tumor Immune Response
1. Introduction
Under normal conditions, cells of the immune system
inhibit tumor growth and progression through the recog-
nition and rejection of malignant cells, a process called
immunosurveillance. However, malignant variants may
create an immunosuppressive microenvironment, block-
ing productive antitumor immunity and resulting in a
shift from immunosurveillance to immunotolerance to a
tumor [1]. Upon metastasis, the biochemical cues se-
creted by tumor cells can directly interfere with the cel-
lular communication necessary for eliciting an appropri-
ate immune response. Tumor induces tolerance by pro-
ducing biochemical cues that suppress immune function,
including TGF-
, IL-4, IL-6 and IL-10 [2,3]. TGF-
in-
hibits the biological activities induced by IL-12 [4]
through an undefined mechanism [5], whereas IL-4 in-
duces polarization towards a Th2 phenotype, promoting
tumor tolerance [6].
Interleukin-12 (IL-12) is an important immune regula-
tory cytokine that exerts potent antitumor activity. At the
cell level, IL-12 is a paracrine cytokine that provides a
critical interface between innate and adaptive immunity
[7]; within secondary lymphoid organs, IL-12 plays a
critical role in promoting antitumor immunity. IL-12 sig-
naling network leads to polarization of naïve CD4 T cells
into a Th1 phenotype [8]. Polarization into a Th1 pheno-
type promotes anti-tumor immunity via cytokine help for
CD8 T cell expansion and switching B cell antibody
production to isotypes, such as IgG2a in the mouse, that
enhance antibody-dependent NK cell-medi- ated cyto-
toxicity [9]. This dual role, as promoter of Th1 polariza-
tion and activator of NK cells, motivates using IL-12 as
an adjuvant for antibody-based tumor immuno- therapy
[10]. IL-12 stimulates IFN
expression, which inhibit
tumor angiogenesis through the interaction among
lymphocyte-endothelial cells, decreasing endothelial cell
survival and adhesion. On the other hand, NK cells,
*We declare no competing interests.
#Corresponding author.
Copyright © 2012 SciRes. JCT
Low Dose of IL-12 Stimulates T Cell Response in Cultures of PBMCs Derived from
Non Small Cell Lung Cancer Patients
338
which are attracted by IL-12, are synergistically citolytic
to endothelial cells.
Lung cancer is a leading cause of cancer death world-
wide. The large majority of cases are non-small-cell lung
cancers (NSCLC), their prognosis is still grim [11] and
novel therapeutic approaches are warranted. Airoldi et al.
demonstrated an anti-tumor activity of IL-12, because it
directly inhibits the growth of human lung adenocarci-
noma and targets the adjacent normal bronchial epithelial
cells [12]. Thus they suggest that IL-12 might be admin-
istered to lung adenocarcinoma patients as tumor-tar-
geted formulations to act directly on the tumor microen-
vironment [13] or systemically to take advantage of the
immunomodulatory activity of the cytokine [14].
After encouraging results in pre-clinical studies, many
Phase I-II trials were initiated with recombinant IL-12 by
intravenous injection [10,15,16] or IL-12 plasmid DNA
by intratumoral injection [17-19], showing some anti-
tumor effects, measurable immunological responses, but
also important dose-dependent side effects.
Recently, evidences of efficacy of low doses of IL-12
on modulation of Th1 vs Th2 has been demonstrated in
an asthma pre-clinical model [20], suggesting a novel
therapeutic approach to diseases which involve a Th1/
Th2 imbalance. In this study Gariboldi et al. tested dif-
ferent doses of IL-12 for the treatment of asthma in mice
and identified 0.01 pg/ml as the lowest and more effect-
tive dose [20].
Considering these last results and the previous clinical
trials, our study aims to investigate the ability of low
doses of IL-12, (in particular 0.01 pg/ml) to modulate the
T cell subpopulations in cultures of PBMCs derived from
patients affected by NSCLC.
2. Material and Methods
2.1. Patients and Cell Cultures
After informed consent, we obtained 20 peripheral blood
samples from patients affected by NSCLC and subjected
to surgical treatment in the Department of Toracic Sur-
gery, A.O. della Salute e della Scienza of Torino. We
isolated peripheral blood mononuclear cells (PBMCs)
from the blood samples after centrifugation over a den-
sity gradient, Lymphoprep (Nycomed Pharma, Norway)
and cultured in α-MEM, supplemented with 10% FBS,
penicillin 100 U/ml and streptomycin 100 μg/m (Cam-
brex, Bio Science, Walkersville, MD), for 10 days.
PBMCs were plated in 24-well plates (2 × 106/well) in
the presence of different concentration of IL-12. All dilu-
tions and preparation of cytokines were prepared starting
from a concentrated solution (1 g/ml) of recombinant
human IL-12 (Sigma-Aldrich, St. Louis, MO). Different
dilution were performed using a 30% hydro-alcoholic
solution as diluent to obtain the following dosages: 10
ng/ml, 1 pg/ml, 0.01 pg/ml. These dosages and the pla-
cebo were tested on the PBMCs derived from all the 20
patients. After 5 days of culture, medium was refreshed
without discarding cells. IL-12 at different doses was
added daily for 9 days.
2.2. Flow Cytometry
To monitor the different number of CD4, CD8 and par-
ticularly of T regulatory and IFN
-producing T cells, we
analysed these sub-populations after PBMC isolation, at
day 5 and 10 of culture.
To perform intracellular staining necessary to detect
the expression of CD4/IFN
, we utilized the BD Cyto-
fix/Cytoperm Fixation/Permeabilization Kit (BD, Bed-
ford, MA), according to the manufacturer’s instructions.
For T regulatory (CD4/CD25high/FoxP3) phenotype ana-
lysis, we used Human Regulatory T cell staining kit
(eBioscience, San Diego, CA). Anti-human PeRCP-CD8
and the relative isotype controls were purchased by BD.
Samples were analyzed in a FACsCalibur instrument
and elaborated by CellQuest software (both from, BD
Bedford, MA). Data represent a percentage of positive
cells, determined by subtracting the percentage value of
the appropriate isotype controls from each sample.
2.3. Cytotoxicity Assay Using the CFSE
Fluorescent-Based Dye
To evaluate the ability of T cells stimulated with low
dose of IL-12 to actually kill cancer cells, we performed
a cytotoxicity assay using carboxy-fluorescein diaceate
succinimidyl ester (CFSE) labelling of target cells H1373,
a human cell line of lung adenocarcinoma (American
Type Culture Center, ATCC). Both PBMCs and H1373
were cultured in RPMI 1640 (GIBCO, Invitrogen, Pais-
ley, UK) supplemented with 10% FBS, penicillin 100
U/ml and streptomycin 100 μg/m (Cambrex, Bio Science,
Walkersville, MD).
H1373 target cells were labelled with CFSE 5 M ac-
cording to manufacturer’s instructions (CellTrace CFSE
Cell proliferation Kit by Invitrogen, Paisley, UK). After
7 days of culture, we collected PBMCs and plated them
on H1373, previously labelled with CFSE, at a ratio 1/10
(i.e. 1 × 105 target cells and 1 × 106 effector cells,
PBMCs) at 37˚C for 24 hours. H1373 were plated alone
with culture media in order to quantify the spontaneous
death, whereas the maximum level of death was obtained
after Triton lysis. The cytotoxic activity of patients’ PB-
MCs was evaluated by flow cytometry based on CFSE
staining of H1373 target cells, and identification of dead
cells by propidium iodide staining of CFSE-labelled tar-
get cells, as previously reported [21]. Acquisition of
Copyright © 2012 SciRes. JCT
Low Dose of IL-12 Stimulates T Cell Response in Cultures of PBMCs Derived from
Non Small Cell Lung Cancer Patients
Copyright © 2012 SciRes. JCT
339
CFSE-labelled H1373 was performed through FAC-
sCalibur instrument (BD Bedford, MA).
thus we studied the Th1 modulation in PBMC culture,
after IL-12 at low doses. At basal level, the mean per-
centage of CD4/IFN
T cells was 30.7%. After 5 days of
culture, CD4/IFN
T cells decreased with 1 pg/ml and 10
ng/ml, whereas it was significantly increased with 0.01
pg/ml (48.4%) and placebo (48.3%). At day 10, we ob-
served a further significant CD4/IFN
T cell stimulation
with 0.01 pg/ml compared to the basal level, (p < 0.01),
whereas there was not a significant difference between
basal condition and placebo (Figure 2).
2.4. Statistical Analysis
The normal distributions of each parameter were deter-
mined by Kurtosis’s test. The number of CD4 and CD8 T
cells were not normally distributed, thus they were com-
pared by means of the Wilcoxon Signed Ranks Test, a
non parametric test for paired samples. Pearson’s corre-
lation coefficients were used to check univariate associa-
tions. The SPSS 17.0 software package was used to pro-
cess the data with p < 0.05 as the significance cut-off. 3.3. Low Dose of IL-12 Increases Lysis of H1373
Cells
3. Results It is known that an increase of IFN-
released by Th1 can
stimulate the activation of cytotoxic CD8 T cells, thus we
evaluated the ability of low doses of IL-12 to induce lysis
of adenocarcinoma cells. Since we observed an increased
number of CD8 T cells after stimulation with 0.01 pg/ml
of IL-12 and a contemporary increase of CD4/IFN-
T
cells, we performed a co-culture of patients PBMCs and
H1373 cells, detecting a significant increase in H1373
lysis with 0.01 pg/ml of IL-12 compared to the placebo
condition, p 0.05 (Figure 3).
3.1. Low Dose of IL-12 Stimulates CD4 and CD8
T Cells in PBMC Cultures
IL-12 is known for its stimulatory effect on CD4 T cells,
thus we analysed the ability of low doses of IL-12 to
modulate CD4 subpopulation in patients’ PBMC cultures.
After PBMC isolation (basal level) from patients’ pe-
ripheral blood, the mean value of CD4 T cells in patients’
PBMC samples was 46.7%. At day 5 of culture, the per-
centage of CD4 T cells increased compared to the basal,
but it was comparable among the different conditions of
stimulation (Figure 1). At day 10 of culture, we observed
that CD4 T cells remained high with 0.01 pg/ml, (p <
0.05), whereas they diminished with 1 pg/ml, 10 ng/ml
and placebo. We observed a significant increase of CD8
T cells after stimulation with 0.01 pg/ml compared to the
basal condition, p < 0.05 (data not shown).
3.4. Low Dose of IL-12 Suppresses T Regulatory
Cells
Since in cancer patients an increase of T regulatory cells
has been documented, we analysed the ability of IL-12 at
different doses to modulate T regulatory in PBMC cul-
tures. The mean value of T regulatory (CD4/CD25high/
FoxP3 positive cells) was 1.7% at the basal level, Figure
4. At day 5 of culture, the percentage of T regulatory did
not significantly increase after IL-12 stimulation, whereas
it increased with placebo. After 10 days of culture, T re-
gulatory showed a trend to decrease, suggesting an inhi-
3.2. Low Dose of IL-12 Increases CD4/IFN
T
Cells
The stimulation of Th1 represents a fundamental step for
an efficient anti-cancer response of the immune system,
Figure 1. The histogram shows the percentages of CD4 T cells at basal level, after 5 and 10 days of culture. The numbers on
the column bars represent the mean value plus standard deviation. At day 5, no significant differences are present among the
different culture conditions. At day 10, the number of CD4 T cells was significantly higher with IL-12 at 0.01 pg/ml than the
basal level, p < 0.05.
Low Dose of IL-12 Stimulates T Cell Response in Cultures of PBMCs Derived from
Non Small Cell Lung Cancer Patients
340
Figure 2. CD4/IFN
producing T cells increased after stimulation with IL-12 at 0.01 pg/ml compared to the basal level, p <
0.01. Moreover, at day 10, CD4/IFN
T cells were significantly higher with 0.01 pg/ml than with placebo, p < 0.05. The num-
bers on the column bars represent the mean value plus standard deviation.
Figure 3. The histogram shows the mean value of H1373 lysis by PBMCs, isolated from 5 NSCLC patients. IL-12 at 0.01
pg/ml enhanced the H1373 lysis induced by PBMCs, p < 0.05.
Figure 4. The histogram shows the modulation of T regulatory cells during culture with and without IL-12. The numbers on
the column bars represent the mean value plus standard deviation. CD4/CD25/FoxP3 was not increased after IL-12 stimula-
tion, at any dosages and they were reduced at day 10 of culture.
Copyright © 2012 SciRes. JCT
Low Dose of IL-12 Stimulates T Cell Response in Cultures of PBMCs Derived from
Non Small Cell Lung Cancer Patients
Copyright © 2012 SciRes. JCT
341
bitory effect of IL-12 on them. In PBMCs treated with
placebo, T regulatory decreased, but they were higher
than at the basal condition, at day 10 (Figure 4).
4. Discussion
Early in vitro studies demonstrated that IL-12 could en-
hance the cytolytic potential of lymphocytes from pa-
tients with a variety of both solid and haematological
malignancies: an overnight incubation with low doses of
IL-12 induced T-cell-mediated lysis of both leukemic
and solid tumor cell lines [22,23]. Based upon encourag-
ing responses in murine models, many clinical trials on
different tumors had been performed, showing somewhat
modest clinical results and, in some instances, untoward
toxicity [15]. Since IL-12 is an important modulator of
immune response and mediates various anti-tumor ef-
fects, we analyzed the ability of low doses of IL-12 to
modulate CD4 subpopulation in cultures of PBMC de-
rived from lung cancer patients. In literature evidences of
efficacy of low doses of IL-12 on modulation of Th1 vs
Th2 has been demonstrated in an asthma pre-clinical
model [20], suggesting the possibility to utilize low doses
of IL-12 to act on other immune pathologies character-
ized by Th1/Th2 imbalance.
Our results show that CD4 T cells proliferate and were
viable in culture, at day 5 with or without IL-12 stimula-
tion, whereas IL-12 at 0.01 pg/ml significantly stimulated
CD4 at day 10, suggesting that this low dose of IL-12
was able to support CD4 proliferation. Doses of IL-12
higher than 0.01 pg/ml and placebo did not show a statis-
tically significant stimulation in CD4 T cells, thus IL-12
at low doses seems to modulate CD4 population. Also
CD8 T cells were increased compared to the basal num-
ber, after stimulation with 0.01 pg/ml of IL-12.
To better define the action of the low dose of IL-12 on
CD4 T cells, we studied the expression of IFN
on CD4
T cells after isolation and IL-12 stimulation. IL-12 at 1
pg/ml and at 10 ng/ml failed to stimulate CD4/IFN
pro-
ducing cells, in particular IL-12 at 10 ng/ml dramatically
down-regulated these cells. IL-12 at 0.01 pg/ml signifi-
cantly stimulated CD4/IFN
producing cells, which were
significantly higher with 0.01 pg/ml than with placebo.
This result demonstrates that IL-12 at 0.01 pg/ml stimu-
lates proliferation of Th1. The increase in CD4/IFN
T
cells is fundamental to activate an anti-cancer response.
Here, we showed an increased lysis of adenocarcinoma
cells after co-culture with patients’ PBMCs stimulated
with 0.01 pg/ml of IL-12 compared to placebo. These
data suggest that this low dose of IL-12 may be effective
for the inhibition of cancer cell proliferation. Since the
inhibitory role of T regulatory cells on immune response
against cancer has been demonstrated [24], we studied
the ability of low doses of IL-12 to down-regulate T
regulatory in PBMC cultures. We showed that the num-
ber of T regulatory was higher with placebo than with
IL-12, which shown an inhibitory effect on T regulatory
cells. These data confirm our hypothesis that IL-12 at
low doses immuno-modulates T cell sub-populations in
vitro, stimulating Th1 and inhibiting T regulatory cells.
Moreover, our results are in accordance with recent pub-
lished data concerning the use of IL-12 at low dose to
treat asthma [16], but need to be also confirmed also on
patients affected by other type of tumors or immu-
nological disorders characterized by unbalance of cyto-
kines and T cell subpopulation.
5. Conclusion
In the present work, we demonstrated that low doses of
IL-12 modulate the T cell subpopulations in cultures of
PBMCs derived from patients affected by NSCLC. We
observed an increased number of CD4 and CD8 after
stimulation with low dose of IL-12, in particular of
CD4/IFN
producing cells, whereas there was an inhibit-
tion of T regulatory cells. This low dose of IL-12 also
promoted the inhibition of the proliferation of lung ade-
nocarcinoma cells in vitro. The possibility to utilize low
dosages of IL-12 as immuno-modulator opens new per-
spectives to study also other cytokines at low doses in
order to restore the balance in the immune response for
diseases characterized by immune system alterations.
Further studies, mainly in vivo, are mandatory to better
characterize the immune effector mechanisms involved
in this model of immunological therapy and to define the
possible use of IL-12 at low dosage to support the im-
mune response in patients affected by NSCLC.
6. Acknowledgements
We thank Compagnia di San Paolo for financial support.
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