| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
|
|
|
|||||||
|
|||||||||
Surgery Branch, Center for Cancer Research, National Cancer Institute, National Institutes of Health, Bethesda, MD 20892
 |
Abstract |
|---|
 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
-chain of IL-2R complex reduced the viability mediated by
IL-2 secretion of the IL-2 transductants. Moreover, transduction of an
IL-2 gene did not affect the high degree of recognition
and specificity of transductants against melanoma targets. These
tumor-reactive IL-2 transductants may be valuable for in vitro studies
and for improved adoptive transfer therapies for patients with
metastatic melanoma. |
Introduction |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
Lymphocyte survival in vivo is dependent on appropriate stimulation, but also on the availability of sufficient local concentrations of appropriate growth factors such as IL-2 (5). To sustain the survival of activated lymphocytes following in vivo administration, it has been necessary to supply an exogenous source of IL-2, although the systemic toxicity of IL-2 severely limits the amount of this cytokine that can be given (6). Although multiple studies of the adoptive transfer of activated lymphocytes with specific function to humans with HIV infection and cancer have been attempted, the limited survival of these cells in vivo has severely compromised their function (4, 7, 8, 9, 10, 11). One potential solution to this problem is the introduction of genes into lymphocytes that can result in either the regulated or constitutive production of appropriate growth signals that might obviate the need for the administration of potentially toxic levels of administered cytokines.
Introduction of an exogenous IL-2 gene to a murine CD4 T cell line led to IL-2 secretion and growth of that cell line independent of added IL-2 in vitro (12). Tumor cells of a variety of histologies when modified by an IL-2 gene could produce IL-2, and such modified cells have been used as tumor vaccines in at least 24 current human gene therapy trials (13). However, there have been no convincing data that IL-2 gene modification can be achieved in human primary T cells, especially CD8+ T cells that are used in adoptive transfer therapy for patients with cancer. One report described the transfection of the IL-2 gene into human tumor-infiltrating lymphocytes (TILs)2 isolated from pleural effusions of advanced lung cancer patients (14), although few data were presented concerning the production of IL-2 and the function of these modified cells.
It is against this background that we undertook studies to introduce an exogenous IL-2 gene into specific tumor-reactive lymphocytes in an attempt to enhance their survival and reduce their dependence on the exogenous administration of IL-2.
|
Materials and Methods |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
To construct a retrovirus to deliver the IL-2 gene to primary T lymphocytes, we used a pSAMIL-2EN plasmid (kindly provided by P. Hwu, Surgery Branch, National Institutes of Health, Bethesda, MD), which contained an IL-2 cDNA A50. A PCR reaction was performed using pSAMIL-2EN as a template to obtain a 490-bp fragment. Primers used to generate this fragment were: 1) 5'-GGAGGCCTGGATCCATGTACAGGATGCAACTCCT-3', and 2) 5'-GGGTCGACGGATCCTCAAGTTAGTGTTGAGATGA-3'. Primer 2 inserted a SalI restriction site (italicized) at one terminus (translational start and stop codons of the IL-2 gene are underlined). This fragment was directionally cloned into the SrfI (Stratagene, La Jolla, CA) and SalI (Promega, Madison, WI) sites of the plasmid pGCIRESYFP (15) (kindly provided by G. Costa, Stanford University, Stanford, CA), resulting in the final bicistronic construct, pIL-2-IRES-YFP, whose structure was confirmed by the nucleotide sequencing from 5' to 3' long terminal repeats. Yellow fluorescent protein (YFP, a variant of enhanced green fluorescent protein) as a marker was used in our study to facilitate the evaluation of transduction efficiency and for positive selection by FACS sorting. The expression of the IL-2 gene in the pIL-2-IRES-YFP is under the viral 5' long terminal repeat promoter control, and the translation of YFP is facilitated by the internal ribosomal entry site (IRES). This obviated the theoretical concern of promoter competition if an internal promoter were used for the expression of the marker gene (12, 17).
Construction of pseudotyped retroviruses
The control vector (IRES-YFP, referred to as YFP) and vector containing the IL-2 gene (IL-2-IRES-YFP, referred to as IL-2YFP) were prepared in parallel. Two packaging cell lines, Phoenix E and PT 67, were grown in DMEM (Life Technologies, Gaithersburg, MD) supplemented with 10% FCS (HyClone Laboratories, Logan, UT) in a 37°C humidified incubator with 5% CO2. Ecotropic Phoenix E was purchased from the American Type Culture Collection (Manassas, VA) with the permission of G. Nolan (Stanford University), and amphotropic PT 67 was purchased from Clontech (Palo Alto, CA). Twenty micrograms of plasmid DNA were mixed with the GeneJammer transfection reagent (Stratagene) and transfected to 2 x 106 Phoenix E cells, according to manufacturer’s instruction. At 48 h post-transfection, the culture medium was used immediately to infect 1 x 107 PT 67 cells in the presence of 8 µg/ml Polybrene. Forty-eight hours postinfection, PT 67 cells were detached from the flask by digestion with trypsin/EDTA (Biofluids, Rockville, MD) and prepared for FACS analysis by a FACScan flow cytometer (BD Biosciences, Mountain View, CA). YFP+ (FITC channel) cells were sterilely sorted twice on a FACSorterPlus (BD Biosciences) (18). The expression of the IL-2 gene from the stable IL-2YFP vector-producing PT 67 line was confirmed by the presence of from 5000 to 8000 IU/ml IL-2 present in the supernatant, as detected by ELISA (Endogen, Woburn, MA). Retroviral titer was determined as described (19). Consistently, supernatants of 0.5–1.7 x 106 TU/ml were obtained.
In vitro stimulation of PBMCs with gp100:209–217 (210 M) (referred to as 209-2M) peptide
Cryopreserved PBMCs obtained after the eighth weekly s.c. injection with 209-2M peptide (IMDQVPFSV) (in IFA) of the patient RP with metastatic melanoma (20) were thawed in complete medium (CM) consisting of RPMI 1640 (Life Technologies) supplemented with 10 mM HEPES buffer, 100 U/ml penicillin and 100 µg/ml streptomycin (Biofluids), 20 µM 2-ME, and 10% heat-inactivated freshly pooled normal human male serum (Biochemed Pharmacologicals, Winchester, VA), and plated at 3 x 106 in 2 ml CM with 1 µM 209-2M peptide. On the next day and every 3 subsequent days, IL-2 (kindly supplied by Chiron, Emeryville, CA) was added to the cultures to a final concentration of 300 IU/ml. Cultures were maintained at the cell density of 0.7–1 x 106/ml. Peptide 209-2M (IMDQVPFSV) is a modified immunodominant epitope from melanoma differentiation Ag, gp100, spanning aa 209–217. This altered peptide with a methionine substituting natural threonine at position 2 (thus referred to as 209-2M) was shown to have much higher degree of recognition in binding to HLA-A2 molecule and more immunogenic than native peptide. CTLs elicited by 209-2M also recognize the native peptide 209-pulsed T2 cells (23).
CD8+ T cell clone (D4F12)
This clone was isolated from a TIL bulk culture obtained from a metastatic lesion of the patient MD, and was kindly provided by M. Dudley and the TIL laboratory (Surgery Branch, National Cancer Institute). The D4F12 clone was grown in 50/50 medium consisting of a 1:1 volume ratio of CM and AIM V medium (Life Technologies) supplemented with 300 IU/ml IL-2. As described below, this clone was expanded by the rapid expansion protocols (REPs) with irradiated (35 Gy) allogeneic feeders and OKT3. On day 7 of REP 3, the transduction was performed.
Transduction
Transductions were performed in wells of 6- or 24-well plates (Becton Dickinson) coated with 10 µg/cm2 Retronectin (Takara, Shuzo, Otsu, Shiga, Japan), according to the manufacturer’s instruction. Two million PBMC or D4F12 cell pellets were resuspended with 8 ml freshly prepared YFP or IL-2YFP retroviral supernatants, respectively. IL-2 was added to YFP supernatants to a final concentration of 7200 IU/ml. No exogenous IL-2 was added to IL-2YFP supernatants, which already contained comparable level of IL-2. This cell-viral supernatant mixture was then applied to a Retronectin-coated well and incubated at 37°C, 5% CO2 for 6 h. A total of four retroviral transductions in the same Retronectin-coated wells was performed in 2 consecutive days. At the end of the second day, the transduced cells were washed twice with CM, resuspended in CM for PBMCs, or 50/50 medium for D4F12, respectively supplemented with 300 IU/ml IL-2. The cultures were maintained at the cell density of 0.7–1 x 106/ml at 37°C, 5% CO2. On day 4–5 posttransduction, transduction efficiency was determined as the percentage of YFP+ cells by a FACScan (FITC channel). These YFP+ cells were sterilely sorted on a FACStarPlus sorter. Sorted transductants were incubated at 37°C, 5% CO2 in CM or 50/50 medium supplemented with IL-2 at a concentration of 300 IU/ml.
Microwell viability and IL-2 production assay
Sorted transductants were washed three times with CM. Cells of
0.5 x 105 in 100 µl CM or 50/50 media
were placed in wells of 96-well U-bottom plate (Costar, Corning, NY)
uncoated or coated with anti-CD3 (OKT3; Orthoclone, Ortho-biotech,
Raritan, NJ) and anti-CD28 (BD PharMingen, San Diego, CA) in the
absence or presence of IL-2 at a concentration of 300 IU/ml. Wells were
coated with OKT3 and anti-CD28, as described (21). As
indicated, anti-CD25 (an Ab reactive with the -chain of the
IL-2R complex, 5–10 µg/ml) or a control isotope Ab (5–10 µg/ml)
(gifts from Y. Tagaya, Metabolism Branch, National Cancer Institute)
was added to the no IL-2 condition. Two days later, 50 µl media were
removed from the wells lacking added IL-2 and assayed for IL-2 by
ELISA. These wells were replaced with 50 µl fresh media without IL-2.
Also on day 2, cells from OKT3- and anti-CD28-coated wells were
transferred to regular wells (nonspecific TCR restimulation for 2 days
only). Fresh IL-2 (300 IU/ml) was added to +IL-2 condition every 3
days. On day 6, the cell clusters at the bottom of all wells were
photographed. On day 7, viable cells were scored using CellTiter 96
Aqueous One solution cell proliferation assay
[3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophe-nyl)-2H-tetrazolium,
inner salt] (MTS); Owen’s reagent) viability assay; Promega],
according to the manufacturer’s instruction. Included in the assay
were standards of viable cells with known numbers, as determined by
trypan blue (BioWhittaker, Walkersville, MD) exclusion, and counted. A
standard curve was generated, and viable cell equivalents were
calculated from this curve.
Rapid expansion protocol (REP)
Cells were expanded, as described previously (8), with minor modifications. A total of 1 x 105 sorted PBMC transductants was added to 25 ml CM without IL-2 in a 25-cm2 tissue culture flask containing 2.5 x 107 irradiated (35 Gy) allogeneic PBMCs and OKT3 at 30 ng/ml. IL-2 was added to some flasks on the next day to a final concentration of 300 IU/ml. Twenty milliliters of media were removed on day 7 and replaced with fresh CM with or without IL-2 (300 IU/ml). On day 9, cells from some flasks were rigorously washed three times and resuspended in CM without IL-2. Viable cells of each condition were counted by trypan blue exclusion, and the total number of viable cells was plotted against time. To test for the existence of an IL-2 autocrine loop, cells from the no IL-2 condition were washed twice with CM and incubated with anti-CD25 or a control isotope Ab at a concentration of 5–10 µg/ml at 37°C, 5% CO2 in the wells of a 48-well plate. Forty-eight hours after incubation, the supernatants were harvested and the IL-2 level was assayed by ELISA. For REPs performed in the wells of 24-well plates (Costar), 1 x 105 effector T cells were incubated with 1 x 106 irradiated (35 Gy) allogeneic PBMCs in 2 ml media containing 30 ng/ml OKT3.
Measurement of melanoma-specific T cell reactivity (20)
A total of 5 x 104 cells of the
sorted transductants or D4F12 was cocultured with 1 x
105 target cells in the microwells of 96 U-bottom
plates in a final volume of 200 µl. Cocultures were incubated at
37°C in 5% CO2 overnight. One hundred thirty
microliters of the supernatant were harvested, and the IFN-
released
by reactive T cells was analyzed by ELISA (Endogen). Target cells used
were T2 cells (HLA-A2+, Tap-deficient line
derived from a B-T cell hybrid (22)) pulsed with peptide
gp100:209–217 (209 peptide) at concentration of 1, 0.01, and 0.0001
µM, respectively; T2 cells pulsed with 1 µM MART-1:27-35 (MART-1);
and melanoma cell lines 526 mel (HLA-A2+) and 888
mel (HLA-A2-). Positive IFN- values were
defined as at least 100 pg/ml and at least twice background.
|
Results |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
Cryopreserved PBMCs obtained from patient RP after eight weekly
injections of 209-2M peptide were thawed, stimulated with the
immunizing peptide, and transduced with retroviruses. To determine an
optimal time point for transduction, a time course of T cell surface
marker and transduction efficiency was investigated (Table I
). At the indicated times after peptide
stimulation, cells were transduced with either YFP control vector or
IL-2YFP vector supernatant on Retronectin-coated plates (21, 31). Four days post-transduction, equal portions of cells were
taken for analysis of T cell surface markers and for transduction
efficiency. As shown in Table I, CD8+ cells
increased from day 6 to day 9 after peptide stimulation (52% to 64%).
A significant increase in transduction efficiency was seen for both
control vector (YFP) and the IL-2YFP vector over time, with the highest
transduction efficiency seen on day 5 after peptide stimulation. The
vast majority of YFP+ cells were
CD8+ (78–96%), as expected by the preferential
proliferation of CTL precursors after a class I-restricted peptide
stimulation. When transduction was performed on day 6 after peptide
stimulation in another independent experiment, the efficiency was 19%
for control YFP vector and 6.3% for IL-2YFP vector, respectively.
Therefore, day 6 postpeptide stimulation was chosen for later
experiments.
|
, suggesting that
the lower efficiency seen in the IL-2YFP transduction was not due to a
deleterious effect of the IL-2 gene expression. IL-2YFP PBMC transductants were able to secrete IL-2 and maintain their viability in the absence of exogenous IL-2 upon restimulation
We next examined the growth and phenotypes of the transduced cells
in the absence of added IL-2 in the tissue culture media. PBMCs were
stimulated with 209-2M peptide, and 6 days later transductions were
performed. The transduction efficiency was determined 5 days later by
scoring YFP+ cells (28.9% for YFP vector and
16.7% for IL-2YFP vector), followed by sterile sorting of
YFP+ cells (Fig. 1). The sorting efficiency for YFP and
IL-2YFP was 93.8% and 76.3%, respectively. These sorted cells were
assayed for their ability to produce IL-2 and for their growth and
viability in microwells of 96 U-bottom well plates.
|
In the absence of IL-2 and no stimulation with OKT3 and anti-CD28,
there was no difference in the size of cell clusters in untransduced or
YFP or IL-2YFP cells (Fig. 2a). Furthermore, all cells
had lost their viability in the MTS viability assay (Fig. 2c). However, with the continuing presence of IL-2 in media,
all groups retained their viability (Fig. 2, b and
d).
|
, e and
g). When IL-2 was present in the media, there were no
differences seen in cell clusters and viability for all transductants
(Fig. 2, b and d, f and h).
Thus, it appeared that the expression of the IL-2 gene by
the IL-2YFP transductants could sustain their survival when
restimulated with OKT3 and anti-CD28 in the absence of added
IL-2.
A second round of stimulation was then performed. The IL-2YFP
transductants that had been stimulated with OKT3/anti-CD28 in the
absence of IL-2 from a replica plate, as shown in Fig. 2e,
were washed twice and subjected to similar restimulation, as described
in Fig. 2. As controls, untransduced and YFP transductants from the
+IL-2 +OKT3/anti-CD28 condition from a replica plate as shown in
Fig. 2f were also restimulated.
As shown in Fig. 3, the same patterns of
results were obtained: only IL-2YFP transductants were viable in the
absence of IL-2 (a and c), and no significant
difference was seen when IL-2 was present in the medium (b
and d).
|
IL-2YFP PBMC transductants were able to proliferate in the absence of exogenous IL-2 upon restimulation
To determine whether the IL-2YFP-transduced cells were capable of
actively proliferating in the absence of IL-2, a REP experiment was
performed. IL-2YFP transductants were obtained from the no IL-2
condition of a second stimulation from a replica plate, as shown in
Fig. 3a. YFP transductants were obtained from a cell culture
that had been maintained after sorting without any restimulation in the
presence of IL-2. Both transductants (1 x
105 each) were washed twice to remove any
residual IL-2 and subjected to the REP protocol with soluble OKT3 and
irradiated allogeneic PBMCs in wells of a 24-well plate with or without
exogenous IL-2 in the culture medium. On day 14 of this experiment,
viable cells were counted by trypan blue exclusion.
YFP transductants did not proliferate at all in the absence of IL. In contrast, IL-2YFP transductants proliferated and expanded 10.4-fold in the absence of IL-2. In the presence of IL-2, YFP transductants expanded 230-fold, and IL-2 transductants expanded 135-fold.
These results established that IL-2-sorted transductants were able to proliferate in the absence of exogenous IL-2 upon restimulation. The difference between YFP and IL-2YFP transductants in the presence of IL-2 might be due to greater growth potential of YFP transductants because they had not been restimulated before this experiment.
Transduction of an IL-2 gene into cells with antitumor reactivity did not affect tumor recognition of transductants
IL-2YFP and YFP transductants, as shown in Fig. 1, were maintained
in the presence of IL-2 after sorting. On day 14 after sorting, they
were analyzed for the release of IFN- in a coculture assay with
tumor targets. Both transductants exhibited high degree of recognition
and specificity for 209 peptide-pulsed T2 cells (Table II, experiment 1). As a further test, the
transduced PBMCs were cloned two days after sorting. The resultant
clones (P209IL-2YFP clone 3 and P209YFP clone 29) were expanded in a
REP protocol in the presence of IL-2. On day 10 of this REP experiment,
an independent coculture assay with the target cells as shown in Table II was performed and the IFN- released by these clones was assayed
by ELISA. These clones showed high degree of recognition and
specificity for 209 peptide and recognized melanoma tumor targets in an
HLA-A2-restricted fashion (Table II, experiment 2). More importantly,
in another independent experiment (Fig. 4, middle), IL-2YFP
transductants that proliferated in the absence of IL-2 specifically
recognized 209 peptide-pulsed T2 cells (Table II, experiment 3). Thus,
transduction of either a reporter gene or an IL-2 with the reporter
gene did not affect tumor recognition of transductants.
|
|
We next examined the viability of IL-2YFP transductants when IL-2
was withdrawn from the culture medium when cells were actively
dividing. To investigate this, an independent transduction experiment
was performed. Bulk PBMCs from patient RP were stimulated with peptide
209-2M, and transduced with IL-2YFP retroviral vector (transduction
efficiency 6.3%) and positively selected on a FACS sorter, as
described. Cells were also transduced with control vector, YFP
(transduction efficiency 19.7%), but not sorted. Cells were washed
twice to remove residual IL-2 and subjected to a REP protocol. Three
conditions were included in this experiment: no IL-2; +IL-2; and +IL-2
until day 9. Cells were counted on the indicated days (Fig. 4) by
trypan blue exclusion.
Upon withdrawal of IL-2 from the medium on day 9, control cells (YFP)
decreased dramatically in their viability 5 days later and there were
no viable cells left at day 25 (Fig. 4, left). In contrast,
IL-2YFP transductants maintained their growth peak for a week before
decreasing in viability after IL-2 withdrawal, and maintained at this
level for another 2 wk before dying. In cultures that underwent a REP
in the absence of IL-2 from the onset of the culture, IL-2YFP
transductants could proliferate in the absence of exogenous IL-2 (Fig. 4, middle). At the peak of their growth, 400-fold expansion
was obtained. Control YFP transductants did not proliferate at all,
confirming the result described earlier. In the presence of IL-2, there
was no difference seen for the growth of YFP transductants when
compared with IL-2YFP transductants (Fig. 4, right).
To extend these results, a second REP was performed. IL-2YFP
transductants that had grown in the absence of exogenous IL-2 were
obtained from the cells shown in Fig. 4, middle, on day 18
(indicated by an empty arrow). Control YFP cells were obtained from the
added IL-2 condition on day 18 (Fig. 4, right, indicated by
a white arrow). The cells were stimulated in a REP experiment (REP2)
using the same conditions as shown in Fig. 4. For this experiment, the
cultures were coded and counted in a blinded fashion. Withdrawal of
IL-2 from the medium on day 9 resulted in a rapid decrease in viability
of YFP transductants, although the IL-2YFP transductants grew and
maintained viability for 3 wk (Fig. 5, left). In contrast, in the complete absence of added IL-2,
IL-2YFP transductants proliferated again, and a higher fold expansion
of IL-2YFP transductants was seen (900-fold at the peak, Fig. 5, middle). This could be attributed to selection of more
IL-2-expressing transductants during the first REP. During the first
REP, IL-2YFP transductants may have been positively selected in the
absence of IL-2. This was supported by the observation that at day 17
of the REP2 culture, in the absence of IL-2, almost all IL-2YFP
transductants were YFP+ cells (thus containing
IL-2 gene), compared with approximately 80%
YFP+ cells on day 13 of REP1 (data not shown).
Furthermore, a longer and a higher level of viability was seen for
IL2YFP transductants after IL-2 withdrawal during the REP2 (compare the
left panels of Figs. 4 and 5).
|
To test the secretion of IL-2 during the peak of their
proliferation and whether an autocrine loop of IL-2 existed by the
IL-2YFP transductants, the following experiment was performed. On day 9
of the second REP from the previous experiment, IL-2 secretion from
cells from IL-2YFP transductants (Fig. 5, middle) in the
absence of IL-2 was compared with control cells from YFP transductants
grown in the presence of IL-2 (Fig. 5, right). Cells were
washed twice to remove IL-2, resuspended in medium without IL-2, and
plated in the presence of IgG2a, anti-CD25, or no Ab. Two days
after incubation, the IL-2 present in the media was assayed by
ELISA.
At the peak of their growth, there was no IL-2 detectable in the medium
from IL-2 transductants, possibly due to active consumption of IL-2 by
these transductants (data not shown). When the -chain of the IL-2R
was blocked by anti-CD25 Ab, 155 ± 3 (SEM) pg/ml IL-2 could
be detected in the medium of IL-2YFP transductants (3.5 x
105 cells in a volume of 500 µl). This
production of IL-2 and its consumption by the same cells indicated the
presence of an autocrine loop. There was no detectable IL-2 in any
condition from YFP transductants (data not shown).
CD8+ T cell clone when transduced with an exogenous IL-2 gene secreted IL-2 and survived in the absence of IL-2
One plausible explanation for the observed phenotype of IL-2 PBMC transductants (IL-2 secretion, active proliferation, and prolonged survival upon IL-2 withdrawal) when compared with control YFP transductants is that some individual cells in the bulk culture of IL-2YFP transductants might have been inherently capable of better growth. To test this possibility, we transduced a CD8+ T cell clone with the IL-2YFP vector and the control YFP vector, respectively. Transductants were sorted by a FACS sorter, and their properties were analyzed in the absence of added IL-2. In this case, all transductants were derived from one clone, and any difference seen between IL-2 transductants vs control YFP vector transductants could be ascribed to the expression of the IL-2 gene. In addition, the protocol developed for the transduction of clones could potentially be useful for clinical application.
Experiments to determine an optimal time to transduce clones showed that on days 7 and 8 of the REP, transduction efficiency was better than on later days (data not shown). Thus, the CD8+ T cell clone, D4F12 reactive with the gp100:209–217 peptide (8), was transduced on days 7 and 8 of REP3. Transduction efficiency was 21.4% for the YFP vector and 10.3% for the IL-2 YFP vector. Six days after transduction, YFP-expressing transductants were sorted and tested for viability and IL-2 production .
Without restimulation, there was no IL-2 detectable in the medium of
either untransduced, YFP, or IL-2YFP transductants (data not shown).
However, upon restimulation with OKT3 and anti-CD28, IL-2
transductants secreted 700 ± 30 (SEM) pg/ml IL-2 (0.5 x
105 cells in 200 µl). No IL-2 was detected in
cultures of untransduced or control vector transductants (Fig. 6a). When assayed for
viability upon restimulation 6 days after being maintained in the
absence of IL-2, IL-2YFP transductants exhibited significantly higher
viability than untransduced or YFP transductants
(p = 0.0249 by Kruskal-Wallis statistical
test). This viability was partially blocked by anti-CD25 Ab,
indicating that IL-2 was responsible for this viability (Fig. 6b). Low viability was seen for untransduced and YFP
transductants presumably due to endogenous IL-2 secretion after
restimulation (8), which could be detected in the presence
of anti-CD25 (data not shown). This low level of viability was also
reversed by anti-CD25. There were no differences observed in cell
viability when IL-2 was present in the media (Fig. 6c).
These results reproduced those previously described for bulk PBMC
transductants.
|
release (872
pg/ml), and did not recognize an irrelevant peptide, melanoma Ag
recognized by T cell-1-pulsed T2 cells (Table III). Specific recognition of melanoma
tumor cell lines was also seen. The transductants recognized HLA
A2+ 526 mel, but not the
A2- tumor cell line (888 mel) (Table III).
|
|
Discussion |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
In this work, we used the 209-2M peptide (23), a specific
melanoma peptide Ag to stimulate PBMCs from peptide-immunized patients
before retroviral transduction. CTL precursors with Ag recognition were
specifically enriched by such a stimulus, as evident by the high
percentage of CD8+ cells that were transduced and
the specific melanoma reactivity of the sorted transductants (Tables I and II). The 10A1 envelope from the PT 67 packaging cells used in this
study may have contributed to this, since it has been shown that
pseudotyped retroviruses expressing this envelope protein have tropism
for CD8+ T cells when compared with other
envelope proteins (16). This is probably related to the
binding of the 10A1 envelope protein to both the A-murine leukemia
virus receptor (Pit2) and the gibbon ape leukemia virus (Pit1)
receptor for cell entry (24). To validate this approach,
bulk-sorted PBMC transductants (YFP and IL-2YFP, respectively) were
cloned using T cell cloning methods described previously (3, 8). Clones with highly avid tumor recognition were screened and
subjected to FACS analyses to score for YFP positivity. It was found
that almost all YFP+ clones were tumor reactive
in an HLA-A2 restriction fashion (data not shown), reinforcing the
notion that peptide-reactive CTLs preferentially proliferated during
peptide stimulation and were subsequently transduced. A similar
approach was used by others to selectively transduce EBV-specific T
cells with genes encoding a selectable marker and HSV thymidine kinase
after PBMC precursors were stimulated with autologous EBV-transformed B
cells (25). The procedure described in this study
represents a novel and efficient way to introduce exogenous genes to
tumor Ag-specific T lymphocytes, and may be useful for cells reactive
with tumor types other than melanoma.
The other major finding of our work was the prolonged viability of IL-2
transductants in the absence of exogenous IL-2. As shown in Figs. 4
(left) and 5 (left), withdrawal of
IL-2 from the culture medium during logarithmic growth resulted in a
drastic decrease in the viability of mock transductants. In contrast,
provision of an exogenous IL-2 gene to these cells maintained their
viability for more than 3 wk in vitro. Moreover, with restimulation,
IL-2 transductants could be actively grown for 8 wk in the absence of
added IL-2 (Figs. 4, middle, and 5, middle). We
attribute this to the existence of an IL-2 autocrine loop; IL-2
transductants constitutively produced IL-2 to support their own growth
and/or survival. This finding could have profound clinical implications
for the development of cell transfer therapies for patients with
cancer. In our prior studies of cell transfer in murine tumor models,
we have shown that the concurrent administration of exogenous IL-2 is
essential for the effective elimination of invasive tumors
(26). Similarly in the human, we have shown that the
survival of transferred lymphocytes may be limited by the inability of
humans to tolerate the high doses of exogenous IL-2 required to sustain
the survival of IL-2-dependent cells (8). The constitutive
IL-2 expression of IL-2 transductants described in this work could
substantially prolong the survival of transferred cells in vivo and
enhance their antitumor activity. In addition, IL-2 produced locally by
these cells at the tumor site might be used by other T cells,
lymphokine-activated killer and NK cells, etc., thus augmenting the
effect. This may limit the need for the administration of high-dose
IL-2 and reduce its toxicities. Further clinical research is planned to
investigate the fate and impact of these IL-2 gene-modified
cells when transferred to patients with metastatic melanoma.
The production of IL-2, and the proliferation and prolonged viability of IL-2 transductants depend on restimulation of these transductants. These results are in agreement with other reports that expression of transgenes in primary T cells is positively correlated with the activation status of the transductants. Pollok et al. (21) described that primary human T cells when transduced with a mB7-1 gene could enhance the transgene expression by repeated stimulation with anti-CD3 and anti-CD28, even at 31 days posttransduction. In a gene therapy feasibility trial for HIV in which a dominant-negative mutant of HIV rev protein was controlled by a retroviral vector, it was shown that only highly activated, transduced cells were resistant to HIV replication (27). The fact that IL-2 expression by transductants depends on restimulation may have in vivo implications; the presence of tumor Ag(s) in the local milieu of the tumor site may serve as a continuous stimulus for IL-2 production, which may ultimately enhance their antitumor activity.
The detectable level of IL-2 produced by IL-2 transductants was limited
(from 155 to 700 pg/ml) possibly due to the active consumption of IL-2
by the cells. The anti-CD25 Ab used in these experiments to block
IL-2 consumption may not have been completely efficient in blocking the
IL-2 autocrine loop of these cells, which were activated by peptide
stimulation, followed by nonspecific TCR restimulation with OKT3 and
anti-CD28. These stimuli are known to up-regulate CD25 expression.
Furthermore, IL-2 itself can induce CD25 up-regulation by binding to
the intermediate affinity 
-chain of the receptor complex
(28). All these factors make it difficult to measure the
true amount of IL-2 produced by IL-2 transductants, although the local
concentration of IL-2 in the surrounding milieu of the lymphocytes in
vivo at the tumor site may be much higher than the amount of IL-2
released into a relatively large culture medium. This low level of IL-2
produced in vitro was reflected in a lower level of growth of these
transductants in the absence of exogenous IL-2, when compared with 300
IU/ml IL-2 present in the medium (Fig. 4, middle vs
right and Fig. 5, middle vs right). In
addition, as shown in Figs. 4, middle, and 5,
middle, IL-2 transductants eventually stopped proliferating
after 16–17 days in cultures not supplied with exogenous IL-2 without
further stimulation. However, with the supplement of 300 IU/ml IL-2,
the same transductants continued to proliferate for 29–35 days. In a
murine model reported by our group, the sperm whale myoglobin-specific
CD4+ T cell line 14.1, retrovirally transduced
with a human cDNA IL-2 gene, remained IL-2 independent
without further stimulation (12). Our results of limited
proliferation of IL-2 transductants are also inconsistent with other
reports of transduction of an exogenous IL-2 gene to murine
T cells. Yamada et al. (29) constructed a pZipSV
retroviral vector containing a human IL-2 cDNA. Transduction
of this vector to an IL-2-dependent murine T cell line, CTLL2, led to
the expression of IL-2. Transductants not only proliferated in vitro in
the absence of IL-2, but also developed tumors (lymphomas) in nude and
syngeneic mice. In another study reported by Karasuyama
(30), a cDNA expression vector BCMGNeo.mIL-2 was
constructed in which a murine IL-2 cDNA was driven by a CMV
promoter. This construct was used to transfect an IL-2-dependent murine
Th cell line, HT-2. It was shown that stable transfectants were able to
secrete high amounts of murine IL-2 and to proliferate autonomously
without exogenous IL-2. Furthermore, high IL-2-producing transfectants
were tumorigenic in nude mice. There are at least two explanations for
the discrepancy between these murine data and the results described in
this study. First, gene expression in mouse cell lines might be
different from that in the normal human primary T cells used in this
study. Second, murine T cell lines used in the above studies might
contain many genetic alterations that may permit higher expression of
IL-2 and might have predisposed them to become easily immortalized and
tumorigenic upon transduction or transfection. Despite these
dissimilarities, it is apparent that opportunities exist to improve
upon expression of the IL-2 gene in our system. These may
include utilization of a different pseudotyping envelope protein, a
different vector, a different promoter to drive the expression of IL-2,
or addition of an IL-2R -chain gene to enhance the impact of the
IL-2 produced. Our current efforts also focus on the transduction of
other cytokine and immortalizing genes that are involved in the
maintenance and survival of lymphocytes into melanoma-reactive human
lymphocytes.
The IL-2 gene-modified lymphocytes described in this study can provide a valuable tool both for in vitro studies and for adoptive transfer therapy to patients with melanoma. The principle developed from this study, i.e., sustaining lymphocyte survival while maintaining their antitumor activity and specificity by gene modification, may be useful for the immunotherapy of patients with cancer and viral infections.
|
Acknowledgments |
|---|
|
Footnotes |
|---|
2 Abbreviations used in this paper: TIL, tumor-infiltrating lymphocyte; CM, complete medium; IRES, internal ribosomal entry site; MTS, [3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium, inner salt]; REP, rapid expansion protocol; YFP, yellow fluorescent protein.
Received for publication July 30, 2001. Accepted for publication September 24, 2001.
|
References |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
This article has been cited by other articles:
|
|
 |
|
  C. Quintarelli, J. F. Vera, B. Savoldo, G. M. P. Giordano Attianese, M. Pule, A. E. Foster, H. E. Heslop, C. M. Rooney, M. K. Brenner, and G. Dotti Co-expression of cytokine and suicide genes to enhance the activity and safety of tumor-specific cytotoxic T lymphocytes Blood, October 15, 2007; 110(8): 2793 - 2802. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
C. Hsu, S. A. Jones, C. J. Cohen, Z. Zheng, K. Kerstann, J. Zhou, P. F. Robbins, P. D. Peng, X. Shen, T. J. Gomes, et al. Cytokine-independent growth and clonal expansion of a primary human CD8+ T-cell clone following retroviral transduction with the IL-15 gene Blood, June 15, 2007; 109(12): 5168 - 5177. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
C. Berger, M. E. Flowers, E. H. Warren, and S. R. Riddell Analysis of transgene-specific immune responses that limit the in vivo persistence of adoptively transferred HSV-TK-modified donor T cells after allogeneic hematopoietic cell transplantation Blood, March 15, 2006; 107(6): 2294 - 2302. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 J. Charo, S. E. Finkelstein, N. Grewal, N. P. Restifo, P. F. Robbins, and S. A. Rosenberg Bcl-2 Overexpression Enhances Tumor-Specific T-Cell Survival Cancer Res., March 1, 2005; 65(5): 2001 - 2008. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 S. A. Rosenberg and M. E. Dudley Cancer regression in patients with metastatic melanoma after the transfer of autologous antitumor lymphocytes PNAS, October 5, 2004; 101(suppl_2): 14639 - 14645. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
C. A. Klebanoff, S. E. Finkelstein, D. R. Surman, M. K. Lichtman, L. Gattinoni, M. R. Theoret, N. Grewal, P. J. Spiess, P. A. Antony, D. C. Palmer, et al. IL-15 enhances the in vivo antitumor activity of tumor-reactive CD8+ T Cells PNAS, February 17, 2004; 101(7): 1969 - 1974. [Abstract] [Full Text] [PDF]
|
|
| ||||||||||||||||||||||||||||
