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Immunology |
Department of Experimental Oncology, Unit of Human Tumors Immunobiology [R. M., A. B., I. B., C. V., A. A.], and Divisions of Pathology [G. T., S. P.] and of Medical Oncology B [E. B.], Istituto Nazionale per lo Studio e la Cura dei Tumori, 20133 Milan, Italy, and Institute of Molecular Medicine, Nuffield Department of Clinical Medicine, Oxford, OX3 9DS, United Kingdom [V. C.]
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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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The efficacy of IL-12 as antitumor cytokine, documented in experimental
models, has opened the way to clinical studies based on systemic
administration of rHuIL-12 to cancer patients (13, 14, 15, 16, 17, 18)
.
The clinical studies conducted thus far have been designed differently
in terms of cytokine dosage (fixed versus escalating dose),
route (i.v. versus s.c.), and schedule of administration, as
well as clinical characteristics of enrolled patients. Despite these
differences among the trials, immunological monitoring has indicated
common effects of IL-12, such as transient lymphopenia (13
, 14
, 16
, 17) and induction of IFN-
in serum (13, 14, 15, 16, 17)
.
In addition, increased levels of IL-10 in serum (14)
and
the enhancement of NK activity and T-cell proliferation in
vitro after therapy have been described (17)
in some
studies. However, it is still unknown whether IL-12 administration in
cancer patients induces systemic effects on T-cell-mediated antitumor
immunity, affects expression of adhesion/homing receptors regulating
T-cell homing/migration, and promotes T-cell infiltration of neoplastic
tissue. To this end, we looked for evidence of modulation of
CTL-mediated antitumor response in vivo in melanoma patients
enrolled in a rHuIL-12 clinical study (14)
. In that study,
10 metastatic melanoma patients received s.c. injections of low-dose
(0.5 µg/kg) rHuIL-12 once a week in three weekly doses (no dose in
week 4) in each of two 28-day cycles. That regimen was well
tolerated by all of the patients, and antitumor activity was clinically
documented, as shown by regression of s.c. nodules, superficial
adenopathies, and hepatic metastases in 3 of 10 patients
(14)
.
As we show here for the first time, rHuIL-12 administration has a systemic impact on T-cell-mediated antitumor immunity, enhances expression of T-cell and endothelial cell adhesion/homing molecules, and promotes infiltration of neoplastic lesions by CD8+ T-cells with a memory phenotype. In addition, in comparison with pretherapy metastases, enhanced tumor necrosis and/or fibrosis was documented in some posttherapy lesions.
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MATERIALS AND METHODS |
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Melanoma Cells.
Melanoma cells were isolated from surgical specimens from 3 of 10
patients enrolled in the rHuIL-12 clinical study and from additional
patients not enrolled in the clinical study. The tumor cells were
established as cell lines and kept in culture as described previously
(19)
. Tumor cells to be used as stimulators and targets
for LDA were cultured in 10% pooled human serum (PHS)-RPMI 1640
(BioWhittaker, Verviers, Belgium). Absence of Mycoplasma
contamination was checked by an ELISA kit (Boehringer Mannheim, Milan,
Italy). Melanoma lines used in this study expressed HLA-class I,
HLA-DR, and -DP antigens and major adhesion molecules
(ICAM-1 and LFA-3).
T-Cell Phenotype.
Lymphocyte phenotype was evaluated after isolation on Ficoll gradients
by means of immunofluorescence followed by flow cytometry analysis
using a FACScan instrument (Becton Dickinson, Sunnyvale, CA). The
following mAbs were used: Leu4 (anti-CD3), WT31 (anti-
ß
TCR) and 11F2 (anti-
TCR; Becton Dickinson). Expression of
adhesion/homing receptors on CD3+ T cells was
evaluated by two-color immunofluorescence. To this end, cells were
first stained with mAbs to CLA (HECA452 mAb, Becton Dickinson), or
integrin 4 chain, or CD44, or CD11a, or CD18 (Immunotech, Marseille,
FR) followed by incubation with FITC-conjugated goat antimouse or
antirat IgG (Jackson ImmunoResearch Laboratories Inc., West Grove, PA)
depending on the primary mAb, and finally by staining with
PE-conjugated anti-CD3 mAbs (Becton Dickinson). Expression of
signal transduction molecules (
chain and lck) in
CD3+ T cells was evaluated by simultaneous
membrane (for CD3 detection) and intracytoplasmic (for chain
and lck detection) immunofluorescence as recently described
(20)
. Statistical comparison of fluorescence histograms
was performed by Kolmogorov-Smirnov statistics.
Cell Cycle Analysis.
Cell cycle analysis in patients’ CD3+ T cells
was carried out by two-color immunofluorescence as described by
Lakhanpal et al. (21)
on cells stained first
with FITC-labeled anti-CD3 mAbs (Becton Dickinson) and then, after
treatment with paraformaldehyde and ethanol, with propidium iodide
(Sigma Chemical Co. LTD., St. Louis, MO). One hundred thousand events
were acquired, and an analysis of cell cycle on
CD3+ cells was performed with the aid of the
Modfit software (Becton Dickinson).
Tetramer Staining.
T-cell staining was performed with PE-conjugated
HLA-A*0201/Melan-A/Mart-126–35
tetramers and HLA-A*0201/influenza
matrix58–66 tetramers. HLA-peptide tetrameric
complexes were synthesized as described previously
(22, 23)
, and specificity of tetramer staining was
reported previously (22, 23)
. Staining of PBLs was
performed by incubating cells for 30 min at 4°C with PE-conjugated
tetramers. As control, two-color fluorescence analysis was performed in
some instances by staining T cells with FITC-conjugated anti-CD8 mAbs
(Becton Dickinson) and PE-conjugated tetramers. The modified
Melan-A/Mart-126–35 peptide (ELAGIGILTV), used
for refolding the HLA-peptide complex has been shown to increase the
binding affinity for HLA-A*0201 without affecting CTL
recognition (24)
. Negative controls for tetramer staining
included PBLs from HLA-A*0201-negative healthy donors,
whereas a Melan-A/Mart-127–35-specific CTL clone
A83 (25)
and fresh TILs, previously shown to
contain a high frequency of
Melan-A/Mart-127–35-specific T cells
(22)
, were used as positive controls. An
influenza-matrix58–66-specific T cell line
(25)
, selected from PBLs after culture for 3 weeks with
peptide-loaded autologous monocyte-derived dendritic cells
(25)
, was used as positive control for staining with
influenza-matrix58–66/HLA-A*0201
tetramers. One x 106 cells for
each PBL sample were analyzed on a FACScalibur instrument (Becton
Dickinson).
Determination of Tumor-specific and Peptide-specific CTLp
Frequency.
Frequency of CTLp directed to autologous tumor or to a peptide from the
melanoma antigen Melan-A/Mart-1
(Melan-A/Mart-127–35; Ref. 26
) was
evaluated by a high-efficiency LDA (22
, 25)
that, as
recently described (22)
, provides frequency estimation in
fresh and activated T-cell populations in the same range as by
HLA-peptide tetramer staining. Split-well analysis for HLA-restricted
precursors was performed at day +28 of culture by comparing lysis of
autologous tumor in the presence or absence of mAbs with CD3 (OKT3,
American Type Culture Collection, Manassas, VA) or to HLA-class I
antigens (w6/32; Ref. 27
). The final cytolytic assay was
performed in the presence of 1.5 x 102 tumor targets/well, and a threshold of 10%
lysis was used. Comparison of two populations of lysis values from the
whole LDA set by ANOVA, followed by the Student-Newman-Keuls
multiple-range test, indicated that a difference between tumor lysis in
the presence or absence of mAbs to CD3 or to HLA-class I 
20 was
significant, with a P 
0.01. By these
criteria, comparison of lysis values in the two aliquots from the same
well allowed the scoring of the original well as containing or not
containing a tumor-specific CTLp (22
, 25)
. The LDA for
detection of peptide-specific CTLp directed to
Melan-A/Mart-127–35 peptide in the context of
HLA-A*0201 were set up in the presence of a constant number
(2000/well) of the TAP-deficient T2 cell line loaded with
Melan-A/Mart-127–35 (AAGIGILTV) peptide (PRIMM
s.r.l., San Raffaele Biomedical Science Park, Milan, Italy) as
described previously (22
, 25)
. Split-well analysis for
peptide-specific CTLp was conducted at day +28 of culture by comparing
lysis of an EBV-transformed, HLA-A*0201+
B-lymphoblastoid cell line (9742 LCL, 3 x 102/well), empty or loaded with
Melan-A/Mart-127–35, as described
previously(22
, 25)
. Thresholds of lysis and criteria to
score a well as containing a Melan-A/Mart-1-specific CTLp were as
described for the LDA sets tested on autologous tumor. A percentage of
negative wells was then evaluated for each lymphocyte dilution. Linear
regression analysis of number of responder cells/well against
logarithmic percentage of negative wells was then performed by using
the Excel software (Microsoft). On the basis of the Poisson
distribution, the slope of the regression line was used to evaluate the
antitumor and peptide-specific CTLp frequency (25)
. To
compare frequency values in distinct LDA sets, the upper and lower 95%
confidence limits of the regression line were evaluated by the Fig.P
software (Biosoft, Ferguson, MO). By this LDA assay, the variation in
CTLp frequency value was less than 15% in independent LDA from the
same blood sample. Furthermore, in control melanoma patients matched
for disease stage but not enrolled in the rHuIL-12 clinical study, the
variation in CTLp frequency value against autologous tumor was less
than 30% in blood samples taken 7 days apart (data not shown).
Detection of Soluble Adhesion Molecules in Serum of Patients by
ELISA.
Serum samples, obtained before and during rHuIL-12 therapy from
patients enrolled in the rHuIL-12 clinical study and from control
patients, were assayed by commercially available ELISA kits (Bender
MedSystems, Vienna, Austria). Presence of the following soluble
adhesion molecules was evaluated: ICAM-1, VCAM-1, and E-selectin.
Results of ELISA experiments were statistically evaluated by ANOVA
followed by the Tukey multiple comparison test.
Immunohistochemical Analysis of Melanoma Lesions.
Immunohistochemical analysis was performed as described recently
(22)
routinely on formalin or on Bouin’s fixed and
paraffin-embedded specimens. To optimize immunodetection of
Melan-A/Mart-1, gp100, CD8, CD45RO, and TIA-1, nonenzymatic antigen
unmasking was performed as described previously (22)
.
Primary antibody incubation was performed overnight at 4°C with the
following antibodies: M2–7C10 (anti Melan-A/Mart-1, Lab Vision Corp.,
Fremont, CA), HMB45 (anti gp-100, Dako Corporation, Carpinteria, CA),
CD8 (Dako), TIA-1 (anticytolytic granule marker, Beckman Coulter,
Fullerton, CA), and CD45RO (Dako). Staining with polyclonal antibody
CD3 (Dako) was performed after 0.1% trypsin treatment for 5 min as
described for antigen unmasking. Sections were subsequently rinsed
three times in PBS + Triton X 100 and treated with
biotinylated goat antimouse immunoglobulin (Dako) or with biotinylated
goat antirabbit immunoglobulin (Dako) for CD3 staining. The slides were
covered with streptavidin-horseradish peroxidase (Dako) for 30 min and
finally visualized with the use of 3-amino-9-ethylcarbazole (Sigma) in
0.05 M acetate buffer containing 0.015%
H2O2. Sections were then
counterstained with hematoxylin and mounted with
glycerin-gelatin 4. Tissue sections subjected to the same
treatment but without incubation with primary antibody were used as
negative controls. A reactive lymph node was used as positive controls
for CD3, CD8, TIA-1, and CD45RO. The positive controls for tumor
antigens were a Melan-A/Mart-1+,
gp100+ lymph-node metastasis of melanoma and a
Melan-A/Mart-1+ human melanoma cell line grown in
nude mice.
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RESULTS |
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, in all of the three patients, a marked and significant increase in
overall tumor-specific CTLp frequency (as evaluated by inhibition of
tumor lysis with anti-CD3 mAbs), as well as in HLA-class-I-restricted
antitumor CTLp frequency (as evaluated by inhibition with w6/32 mAbs)
was observed between 4 and 7 days after the first rHuIL-12
administration. In patient 5, tumor-specific CTLp frequency values
peaked at day +4, after the first rHuIL-12 administration, and dropped
below threshold of detection at day +7 (Fig. 1)
. In blood samples taken
1 and 7 days after the last rHuIL-12 administration (patients 5 and 10,
Fig. 1
), frequency of CTLp inhibited by anti-CD3 or by anti-HLA class-I
mAbs was again higher than in pretherapy PBLs, although lower than the
highest values seen between day +4 and +7 of the first cycle. CTLp
determination in patient 6 could be performed in a blood sample taken
even 1 day after the first rHuIL-12 administration, and at that time,
frequency of CTLp inhibited by anti-CD3 mAbs showed a drop in
comparison with pretherapy values (day -4) and to the subsequent burst
(day +7).
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In two patients (patients 1 and 3) expressing the restricting element
(HLA-A*0201) for the tumor antigen
Melan-A/Mart-127–35, the frequency of CTLp
directed to this immunodominant epitope was evaluated in PBLs by LDA.
In comparison with pretherapy values (day -4) in both patients (Fig. 2)
, a marked increase in peptide-specific CTLp in peripheral blood was
observed between day +4 and day +7 after the first rHuIL-12
administration, as well as between 1 and 7 days after the last rHuIL-12
injection. To corroborate the LDA results, T cells from patient 1 were
stained with
HLA-A*0201/Melan-A/Mart-126–35
tetramers or, as control, with
HLA-A*0201/influenzamatrix58–66
tetramers (Table 1)
. Analysis of day -4 and day +7 PBLs from patient 1 (Table 1)
did not
detect T cells staining with
HLA-A*0201/Melan-A/Mart-126–35
tetramers, in agreement with LDA (<5 CTLp/106
PBLs; see Fig. 2
). However, in day +1 PBLs, the T cells staining with
HLA-A*0201/Melan-A/Mart-126–35
tetramers T cells were 252 CTLp/106 PBLs
and at day +4 tetramer+ T cells were 239
CTLp/106 and 255
CTLp/106 lymphocytes (in two distinct
experiments) in good agreement with LDA data in patient 1 (200
CTLp/106 PBL at day +4; see Fig. 2
). By
contrast, staining of patient-1 PBLs with a control tetramer
(HLA-A*0201 complexed with influenza
matrix58–66 peptide) revealed a reduction in
frequency of influenza-matrix-specific T cells at days +1 and +4 in
comparison with day -4, followed by a rebound at day +7. Control
experiments, in HLA-A*0201+ patients
matched for disease stage but not enrolled in the rHuIL-12 study,
indicated that the frequency of T cells staining with
HLA-A*0201-Melan-A/Mart-1 tetramers was constant in
independent blood samples taken weekly during 1 month (data not shown).
Thus, the experiments with HLA-A*0201-Melan-A/Mart-1
tetramers confirmed the LDA data and indicated that rHuIL-12
administration transiently boosts the frequency of peptide-specific
CTLp directed to a melanoma antigen peptide in peripheral blood.
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ß or TCRs (data
not shown). Furthermore, the expression of two TCR signaling
molecules ( chain and lck) that can be defective in cancer
patients was normal in the treated patients and did not change after
rHuIL-12 administration (data not shown). In addition, cell cycle
analysis of patients’ T cells in PBLs did not show any difference in
CD3+ T-cell proliferation in posttherapy (day +4
and +7) samples in comparison with pretherapy (day -4) T cells in any
of the patients (see Fig. 3
for representative data from one patient).
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4 subunit of VLA-4
(CD49d), as well as a clear increase in fluorescence intensity for
CD44, was observed (Fig. 4)
. The increased expression was transient as, at day +7,
fluorescence intensity for CD49d and CD44 dropped to values lower than
those seen in pretherapy samples. In most patients a new transient
increase in expression of LFA-1, VLA-4, and CD44 on circulating T cells
took place between 1 and 4 days after the last rHuIL-12 administration
of the second cycle (data not shown). In addition (see Table 2
), a clear increase in the proportion of T cells expressing CLA, the
skin homing receptor, was documented, mainly after the first rHuIL-12
administration.
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. In many instances, additional increases took place even at day +4 and
+7 (Fig. 5)
. Soluble endothelial cell adhesion molecule levels declined
after the first cycle of rHuIL-12 administration, but a new increase
was detected in all of the patients at the second cycle (data not
shown). Control experiments were performed by monitoring for 1 week the
serum samples taken from 10 metastatic melanoma patients matched for
disease stage with the patients receiving rHuIL-12. These experiments
indicated that sICAM-1, sE-selectin, and sVCAM-1 levels were either
constant or showed a maximum range of fluctuation within ±30% of the
initial value (data not shown).
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and exemplified in Fig. 6A
(lesion 1 of patient 10), none of the five
pretherapy lesions from the three patients contained infiltrating
CD3+ T cells and, thus, were classified as
"absent." In contrast, from the same patients, 8 of 8 posttherapy
metastatic lesions contained infiltrating T cells with either a
"brisk" or a "non-brisk" pattern (Table 3)
. None of the
posttherapy s.c. lesions showed an enhanced infiltration of T cells in
the areas of normal skin adjacent or overlaying the tumor tissue (data
not shown). In addition, as shown in Table 3
(and exemplified in Fig. 6B
; lesion 2, patient 10), all of the T cells infiltrating
the posttherapy lesions were CD8+ and expressed a
memory phenotype (CD45RO+). Furthermore, a
proportion between 10 and 100% of the T lymphocytes that infiltrated
the lesions expressed the cytolytic granule-associated protein
recognized by the TIA-1 antibody (Ref. 29
; Table 3
).
Histological evidence of increased tumor necrosis and/or regression was
documented in posttherapy lesions from two patients (patients 10 and
2). In these instances, tumor necrosis areas were of the coagulative
type, as defined by nuclear loss and marked cytoplasmic eosinophilia.
In patient 2, histological evidence of tumor necrosis was observed only
in posttherapy lesions (Table 3)
, whereas in patient 10, posttherapy
samples showed larger areas of tumor necrosis in comparison with the
pretherapy metastasis (Table 3
; Fig. 6B
) and presence of
fibrosis in two lesions (lesions 2 and 4 in Table 3
). In patient 3, no
differences in the extent of tumor necrosis were observed between pre-
and posttherapy lesions. Interestingly, none of the lesions in
this HLA-A*0201+ patient expressed two tumor
antigens (Melan-A/Mart-1 and gp100; Table 3
) that can be recognized by
HLA-A2-restricted CTL, and the posttherapy lesion expressed HLA-A2 on
only 30% of tumor cells (as evaluated by immunohistochemistry, data
not shown). This suggested a possible mechanism of immune evasion,
despite the non-brisk T-cell infiltrate found in the posttherapy lesion
of this patient.
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DISCUSSION |
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The rHuIL-12 effect on CTLp frequency was not attributable to a change
in the proportion and phenotype of circulating
CD3+ T cells. In fact, pretherapy values of
circulating CD3+ T cells expressing either ß
or TCRs indicated no significant differences in comparison with
values detected at day +4 or +7, at times when LDA revealed a marked
increase of tumor-specific CTLp. Furthermore, expression of two TCR
signal transduction molecules ( chain and lck), which can be
defective in cancer patients (30, 31)
, was found to be
normal in the enrolled patients, and rHuIL-12 treatment did not improve
the expression of such molecules. Reversal of T-cell anergy by rHuIL-12
is another possible mechanism that may underlie the CTLp burst. IL-12
has indeed been shown to reverse antigen-specific T-cell anergy
in vitro and in vivo (32, 33, 34)
.
However, this mechanism is not supported by the data obtained with
tetramers. The tetramer experiments, with HLA-A*0201
complexed with Melan-A/Mart-126–35,
indicated that an increase in the number of circulating
peptide-specific CTLp did take place, as a result of rHuIL-12
administration, as early as 24 h after the first injection, which
suggests rapid recruitment of tumor-specific CTLp into the blood as a
possible mechanism of the observed burst.
Interestingly, rHuIL-12 administration did not similarly affect the peripheral frequency of any antigen-specific T-cell precursor. In fact, in patient 1, in contrast to the burst observed for Melan-A/Mart-1-specific precursors, the number of circulating influenza-matrix58–66-specific precursors dropped at day +1 and +4 after rHuIL-12 and showed a rebound only at day +7. In addition, evaluation of the frequency of Melan-A/Mart-1-specific T cells in peripheral blood by LDA and by tetramers provided similar values, in agreement with our recent results that indicated that the LDA technique, as modified by us, achieves the same efficiency in detecting antigen-specific T cells as the enzyme-linked immunospot or tetramer staining (22) .
Administration of rHuIL-12 may promote rapid clonal expansion of tumor-specific CTLp. This possibility is supported by several lines of evidence. For example, repeated s.c. injections of IL-12 can lead to selective expansion of a CD8+ T-cell subset characterized by high expression of CD18 and by non-MHC-restricted cytotoxicity after culture with IL-12 plus IL-2 (35) . In mice, the in vivo injection of tumor cells producing IL-12 has been shown to induce proliferation of CD8+ T cells with a memory phenotype (CD44hi) in both spleen and lymph nodes (36) . Furthermore, an increase in the frequency of antitumor CD8+ T cells, as inferred by TCR repertoire analysis, has been suggested in both blood and tumor site after local IL-12 gene therapy in mice (37) . More recently, IL-12 has been shown to act as a "third signal" (in addition to TCR engagement and costimulation) promoting activation and clonal expansion of naive CD8+ T cells (38) . However, in our experiments of cell cycle evaluation, performed as early as 4 and 7 days after the first rHuIL-12 administration, no evidence was obtained of enhanced T-cell proliferation in vivo in peripheral blood. Nevertheless, we cannot rule out the possibility that rHuIL-12 did promote rapid CTLp proliferation in the tissues other than blood and before the peripheral burst of CTLp was induced and detected.
Serum levels of soluble adhesion molecules and phenotype analysis of circulating CD3+ T cells provided evidence for an early and marked effect of rHuIL-12 on several receptor/ligand pairs involved in regulating interaction of T cells with endothelial cells and T-cell homing. The data suggested that s.c. rHuIL-12 treatment could activate a mechanism that may promote adhesion and then extravasation of activated/memory T cells by the LFA-1/ICAM-1, VLA-4/VCAM-1 and CLA/E-selectin pathways. This possibility is in agreement with experimental models that show that IL-12 therapy can induce T-cell infiltration of tumor tissue associated with enhanced VCAM-1 expression on tumor blood vessels (7) . Furthermore, in mice, IL-12-induced T-cell migration to tumor tissue can be inhibited by treating tumor-bearing animals with antibodies to either VLA-4 and LFA-1 (39) . Additional experimental evidence indicates that IL-12 can promote adhesion-dependent homing of T cells to both skin and liver (40, 41) , and in vivo treatment with antibodies to VCAM-1 can abrogate hepatic recruitment, in mice, of T and NK cells induced by systemic IL-12 (41) .
An increased proportion of CLA+ T cells was found in peripheral blood after rHuIL-12 administration. Interestingly, IL-12 has been shown to induce expression of this homing receptor (42) , and it is known that trans-endothelial migration of CLA+ T cells requires the VLA-4/VCAM-1 and LFA-1/ICAM-1 adhesion pathways (43) . Inasmuch as circulating CLA+ T cells comprise memory/effector T cells (44) , then it is possible that rHuIL-12 treatment may promote adhesion-dependent extravasation of CLA+-activated/memory T cells. This possibility is in agreement with the memory phenotype (CD45RO+) expressed by T cells infiltrating posttherapy lesions in the present study. Furthermore, the increased expression of CLA antigens was more pronounced after the first cytokine injection than after the last one. This reduced biological effect of the last rHuIL-12 administration was observed also when looking at CTLp frequency and may be attributable to a reduced systemic availability of the cytokine, as previously described in these patients at the end of the therapy cycles (14) .
The immunohistochemical analysis of metastatic tissues that were removed from patients indicated the presence of a brisk, or non-brisk infiltrate of CD8+ T cells with a cytolytic (TIA-1+) and memory (CD45RO+) phenotype in posttherapy lesions. In addition, although all of the clinically regressing lesions could not be analyzed, other available posttherapy metastases showed increased histological evidence of tumor necrosis and/or regression in comparison with pretherapy tumor tissues. These data suggest that the antitumor activity of rHuIL-12, as evaluated at the histological level, may be greater than previously assessed at the clinical level in the same patients (14) . As suggested by some experimental models (45) and by clinical data obtained in cutaneous T-cell lymphoma patients treated with rHuIL-12 (18) , it is possible that the infiltrating CD8+, TIA-1+, CD45RO+ T cells may be involved in the observed tumor regressions. However, we cannot exclude the possibility that additional mechanisms, such as inhibition of angiogenesis and/or induction of endothelial wall injury, may contribute to the antitumor activity of rHuIL-12 in the clinical setting.
Finally, the increase in peripheral antitumor CTLp frequency was
transient and subjected to attenuation after the last rHuIL-12
administration, in comparison with the peak value detected after the
first injection. A similar "adaptive response," was previously
observed by us and others when looking at the serum levels of IL-12 and
at induction of serum IFN- (13, 14)
and IL-10
(14)
after rHuIL-12 administration. In particular, in
patients, a burst of IFN- was detected in serum within 1–2 days
after the first rHuIL-12 injection, whereas lower levels were induced
after the last rHuIL-12 administration (14)
. The adaptive
response could reduce the toxic effects associated with prolonged IL-12
therapy, but it might also contribute to reducing the antitumor
efficacy of the cytokine. In support of this possibility, it has been
shown (16)
that a single IL-12 dose, given weeks before
consecutive doses, can abrogate IL-12-associated toxicity but can also
can block induction of IFN-, an important mediator of IL-12
biological effects (1)
. At least two mechanisms have been
described that could explain the adaptive response. On one hand, IL-12
administration to either cancer patients or mice, by fixed weekly
doses, promotes enhanced IL-12 receptor expression in lymphoid cells,
which may favor increased IL-12 clearance but leads also to reduced
IFN- mRNA expression (46)
. On the other hand, murine
models of tumor immunotherapy have indicated the involvement of nitric
oxide, produced by macrophages in response to IFN-, in the immune
suppression induced by IL-12 given i.p. (47)
. Additional
studies are clearly needed to fully understand the mechanism of the
adaptive response, thus improving the clinical efficacy of rHuIL-12 in
anticancer therapy.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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1 This work was supported in part by funds from
the Italian Association for Cancer Research (AIRC, Milan, Italy) and
the Italy-United States Program on Therapy of Tumors (Istituto
Superiore di Sanità, Rome, Italy). 
2 To whom requests for reprints should be
addressed, at Human Tumors Immunobiology Unit, Department of
Experimental Oncology, Istituto Nazionale per lo Studio e la Cura dei
Tumori, Via Venezian 1, 20133 Milan, Italy. Phone: 39-02-2390817; Fax:
39-02-2390630; E-mail: Anichini{at}istitutotumori.mi.it
3 The abbreviations used are: IL, interleukin;
CLA, cutaneous lymphocyte antigen; CTLp, CTL precursor(s); ICAM,
intercellular adhesion molecule(s); LFA, lymphocyte function-associated
antigen; LDA, limiting-dilution analysis; mAb, monoclonal antibody;
Melan-A/Mart-1, melanoma antigen-A/melanoma antigen recognized by T
cell; PBL, peripheral blood lymphocyte; rHu, recombinant human; TCR, T
cell receptor; TIL, tumor-infiltrating lymphocyte; VCAM, vascular cell
adhesion molecule(s); VLA, very late antigen; NK, natural killer; PE,
phycoerythrin; FL, fluorescence.
Received 12/28/99. Accepted 4/26/00.
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REFERENCES |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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J. E. A. Portielje, C. H. J. Lamers, W. H. J. Kruit, A. Sparreboom, R. L. H. Bolhuis, G. Stoter, C. Huber, and J. W. Gratama Repeated Administrations of Interleukin (IL)-12 Are Associated with Persistently Elevated Plasma Levels of IL-10 and Declining IFN-{gamma}, Tumor Necrosis Factor-{alpha}, IL-6, and IL-8 Responses Clin. Cancer Res., January 1, 2003; 9(1): 76 - 83. [Abstract] [Full Text] [PDF]
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L. Cifaldi, E. Quaglino, E. Di Carlo, P. Musiani, M. Spadaro, P. L. Lollini, S. Wolf, K. Boggio, G. Forni, and F. Cavallo A Light, Nontoxic Interleukin 12 Protocol Inhibits HER-2/neu Mammary Carcinogenesis in BALB/c Transgenic Mice with Established Hyperplasia Cancer Res., April 1, 2001; 61(7): 2809 - 2812. [Abstract] [Full Text]
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