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*
Department of Immunology, The Scripps Research Institute, La Jolla, CA 92037; and
Lexigen Pharmaceuticals, Lexington, MA 02173
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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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We focused our efforts primarily on an alternative approach to direct cytokines, such as IL-2, to the tumor microenvironment with recombinant Ab-cytokine fusion proteins (immunocytokines), which take advantage of the paracrine nature of most cytokines that function best within a few cell diameters from their cell of origin (6). We previously reported that targeted IL-2 therapy with such immunocytokines induced tumor-specific CD8+ T cell responses that were effective in eradicating established metastases of murine melanoma (7) and colon carcinoma (8) in syngeneic mice. This tumor eradication was followed by a long-lived tumor-protective immunity sufficiently effective to reject a secondary tumor cell challenge in 50% of mice that had been previously cured of experimental pulmonary metastases by Ab-IL-2 fusion proteins (9, 10). Importantly, in the colon carcinoma model, this long-lived tumor-protective immunity was successfully amplified to become optimally effective in 100% of experimental animals by injecting two noncurative doses of Ab-IL-2 fusion protein 4 days after the secondary tumor cell challenge (10).
The persistence of tumor-protective immunity up to 6 mo (10) indicated a long-lived T memory cell response. In this regard, it is well known that primary responses to T-dependent Ags are followed by long-lived immunological memory with the host giving heightened responses following secondary contacts with Ag (11, 12, 13, 14, 15). Long-term memory has been well documented at the T and B cell levels and reflects the combination of increased frequency of Ag-specific precursors and preferential survival of high-affinity cells (15).
Memory cells are thought to appear during the later stages of the primary immune response, and the majority of T lymphocytes participating in this response are short-lived and rapidly eliminated at the end of the response (15). In fact, memory cells were suggested to arise from precursor cells that avoid contact with high concentrations of Ag (16). The notion that low-level stimulation of T cells favors survival rather than death suggests that maintenance of long-lived T cell memory may require continuous exposure to residual Ag depots (15). This idea is supported by reports of poor memory cell survival on adoptive transfer unless accompanied by specific Ag (16). However, the notion that CD8+ T cell memory cells require constant Ag stimulation was challenged by several reports indicating that such cells can survive for prolonged periods after adoptive transfer in the apparent absence of Ag (17, 18).
Here, we examine some of the cellular mechanism(s) involved in these events in our tumor model with emphasis on T cell memory reflected, in part, by specific fusion protein-induced increases in the frequency of precursor CTL (pCTL).3 Treatment of tumor-bearing BALB/c mice with huKS1/4-IL-2 fusion protein increases frequency of pCTL concomitant with the induction of genes encoding primarily Th1 cytokines. Subsequent generation of tumor-specific CD8+ T effector cells is followed by differentiation into long-lived T memory cells, which can be maintained in the absence of tumor Ags.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Female BALB/c and scid/scid mice were obtained from The Jackson Laboratory (Bar Harbor, Maine). The mice were maintained under specific pathogen-free conditions and used for experiments when 7 wk old. All experiments were performed according to the National Institutes of Health Guidelines for the Care and Use of Laboratory Animals.
CT-26, a colon epithelial tumor cell line, derived by intrarectal injection of N-nitroso-N-methylurethane in BALB/c mice, was kindly provided by Dr. I. J. Fidler (MD Anderson Cancer Center, Houston, TX). The KS Ag (KSA) is an epithelial cell adhesion molecule recognized by mAb KS1/4 (19) that was cloned by several investigators (20, 21). KSA was cloned by PCR and expressed in CT26 colon carcinoma cells using a retroviral vector. A subclone of CT26-KSA stably expressing both KSA and MHC class I Ags H-2Kd, H-2Dd was selected for the studies described in this report. Expression of KSA remained stable in culture and on CT26-KSA cells propagated as tumors in BALB/c mice, as indicated by FACS analyses (8). CT26 and CT26-KSA cells were maintained in DMEM, supplemented with 5% FBS, vitamins, L-glutamine, sodium pyruvate, and nonessential amino acids at 37°C and 7.5% CO2. Recombinant human IL-2 (rhIL-2) was obtained from Chiron (Emeryville, CA) with a sp. act. of 16 x 106 IU/mg. All reagents were free of endotoxin, as determined by the Limulus amebocyte lysate assay purchased from BioWhittaker (Walkersville, MD).
Abs and fusion proteins
The generation of the murine KS1/4 Ab recognizing KSA was described previously (19), as was the subsequent construction of the recombinant humanized huKS1/4-IL-2 fusion protein (22). The huKS1/4-IL-2 fusion protein and mAb huKS1/4 revealed identical binding patterns with CT26-KSA cells with an average Kd of 1.15 nM and recognized 3.8 x 106 KSA binding sites/cell (8). The concentration of IL-2 in the huKS1/4-IL-2 fusion protein was calculated as two molar equivalents of IL-2 per mole of fusion protein.
Induction of experimental metastases
Pulmonary metastases in syngeneic BALB/c mice were induced following i.v. injection of 5 x 104 CT26-KSA cells. After 4 days, microscopically established metastases were present throughout the lung tissue. Grossly visible metastases were detectable on the surface of the organs 28 days after tumor cell injection, at which time, the animals were sacrificed and examined for metastases. Lungs were placed in Bouin’s fixative and examined under a low magnification microscope for tumor foci on the surface of the lungs. Since such foci appeared fused, metastases were scored according to the percentage of lung surface involvement with 0, 0%; 1, <5%; 2, 5–50%; and 3, >50% (8). Lung specimens of these mice without macroscopic metastases were stained with hematoxylin/eosin and examined histologically. Some of the samples were snap frozen in -70°C for subsequent RNA isolation.
Tumor cell challenge models
Tumor cell challenges were performed in two different experimental models. First, BALB/c mice, with established pulmonary metastases 4 days after i.v. injection of 5 x 104 CT26-KSA colon carcinoma cells, were administered daily i.v. injections (15 µg each) for 7 consecutive days. These mice, cured of pulmonary metastases, were challenged after 6 wk with 1.5 x 104 CT26-KSA cells, sacrificed 12 days thereafter, and analyzed for frequency of pCTL and for cytolytic activity of T effector cells. Second, the same treatment regimen was used except CD8+ T cells were purified from splenocytes of BALB/c mice obtained 12 days after tumor cell inoculation and 1 day after completion of the 7-day treatment with huKS1/4-IL-2 fusion protein. However, in this case, the purified CD8+ T cells were adoptively transferred to syngeneic SCID mice and parked there for 6 wk, a time after which these mice received a tumor cell challenge, followed 4 and 6 days thereafter by two boosts with noncurative doses (5 µg each) of either huKS1/4-IL-2 or a nonspecific fusion protein, ch14.18-IL-2. Additional control animals received no boost.
Multiprobe RNase protection assay
The expression of total murine cytokine RNA by naive mice and
mice treated with either PBS or KS1/4-IL-2 fusion protein was
determined with an RNase protection assay (RPA; PharMingen, San Diego
CA), which allows for the simultaneous quantification of several mRNA
species in a single sample of total RNA. The murine RPA probe sets used
analyzed for mRNA of murine cytokines IL-2, IL-4, IL-10, IL-6, IFN-
,
GM-CSF, lymphotoxin (LT)-ß, and TNF-
and a housekeeping gene
transcript, L32. The latter encodes a ribosomal structural protein and
facilitates assessments of total RNA levels for normalization of
sampling or technical errors from each sample. Standard precautions
were taken to avoid RNase contamination during all steps of the test.
The probe synthesis uses [-32P], GACU
nucleotide pool and a set of templates supplied by the manufacturer
(PharMingen). Sample RNA of high quality and purity was used to
hybridize with the labeled probe, and 10 µg RNA was added to each
tube, including one containing yeast total RNA as a background control.
Following probe synthesis and overnight hybridization, the RNase
treatment, purification of the protected probe, electrophoresis on
denaturing polyacrylamide gel, autoradiography, and phosphorimaging
were done according to the manufacturer’s guidelines.
Cytotoxicity of CD8+ T effector cells
Splenocytes were isolated from either BALB/c mice or syngeneic SCID mice. To this end, splenocytes were labeled with paramagnetic anti-CD8+ MicroBeads and separated with Mini MACS Separation Unit (Miltenyi Biotec, Auburn, CA) or T cell purification kits (R&D Systems, Minneapolis, MN) according to the manufacturer’s guidelines. Purity of CD8+ T cells was >95%, as determined by FACS analysis. CD8+ T cells were incubated for 3 days at 37°C in 25 ml complete DMEM culture medium containing 1 ml of T-STIM culture supplement (Becton Dickinson, Bedford, MA).
Tumor-specific cytotoxicity was measured in a standard 51Cr-release assay. Briefly, CT26-KSA tumor target cells (3 x 106) were each labeled with 0.5 mCi of 51Cr for 2 h at 37°C, washed three times in DMEM and aliquots of 5 x 103 labeled cells were added to each well of a U-bottom microfilter plate and incubated with effector cells at different E:T ratios at 37°C for 4 h. The percentage of specific target cell lysis was calculated by using the formula: [(E - S)/(T - S)] x 100, where "E" is the average experimental release, "S" is the average spontaneous release, and "T" is the average total release of 51Cr.
Analysis of pCTL frequency
The frequency of pCTL was determined by a limiting dilution analysis. Briefly, spleen cells were harvested from tumor-bearing BALB/c mice on days 0, 7, 12, and 24 of treatment with hKS1/4-IL-2 fusion protein and from a control group treated only with PBS. These cells were diluted serially and cultivated in 98-well flat-bottom plates in the presence of T cell medium and incubated with irradiated (12,000 rads) feeder cells consisting of 1 x 104 cells each of splenocytes from naive BALB/c mice and CT26-KSA colon carcinoma cells. After 5 days, each well was assayed for cytotoxic activity against CT26-KSA target cells. Cultures were designated positive when specific lysis was three SDs above the lysis found in negative controls. The fraction of negative cell cultures was determined for each dilution with 48 cultures per dilution. The frequency of pCTL was assessed, as described previously (23). Briefly, the fraction of negative cultures was plotted on a semilogarithmic scale against the number of splenocytes per well. Frequencies of pCTL were defined by the slope of the linear regression between at least three separate data points. The formula used was lny = (-fx + lna), where "f" is the frequency of responding precursors in a given cell population, "x" is the number of splenocytes added to each culture, "a" is the y-axis intercept (in our experiments, 100% ± 6%) and "y" is the percentage of negative cultures.
Cytokine release assay
Splenocytes were collected from the experimental groups of
BALB/c scid/scid mice cured of pulmonary colon carcinoma metastases by
huKS1/4-IL-2 fusion protein, challenged after 6 wk with CT26-KSA tumor
cells, and boosted 4 days thereafter with either noncurative doses
(2 x 5 µg) hKS1/4-IL-2 fusion protein or with PBS. Lymphocytes
were isolated on Hypaque-Ficoll (BioWhittaker) and cultured for 4, 12,
24, and 48 h in DMEM medium. Supernatants were harvested, obtained
from each group of mice at these time points, and assayed for murine
IFN-, GM-CSF, and IL-2, respectively, with commercially available
cytokine detection kits (BioSource International, Camarillo, CA) using
a solid-phase sandwich ELISA.
Adoptive transfer of lymphocytes
Mice that were successfully treated with huKS1/4-IL-2 fusion protein, which eradicated pulmonary metastases of CT26-KSA tumor cells, served subsequently as donors of lymphocytes for adoptive transfer experiments. These animals were sacrificed 12 days after tumor cell inoculation, followed by the preparation of single cell suspensions obtained by mincing freshly resected spleens and passing them through a steel sieve. Lymphocytes were separated by Ficoll/Hypaque gradient centrifugation (600 x g, 20 min). Subsets of CD8+ T cells were isolated at >95% purity by magnetic-activated cell sorting (Miltenyi Biotec). Naive, syngeneic SCID mice were reconstituted with a total of 4.5 x 107 CD8+ T cells by i.v. injection of 1.5 x 107 CD8+ T cells on days -3, -1, and +3, and their fate was followed by flow cytometry analyses at 1, 2, 3, 4, and 5 wk using anti-CD8+ and anti-CD3+ Abs, respectively. After 6 wk, individual groups of mice were challenged by i.v. injection of 5 x 104 CT26-KSA cells to initiate pulmonary metastases, followed by a boost of two noncurative doses of huKS1/4 fusion protein (2 x 5 µg) on days 4 and 6 after CT-26KSA tumor cell challenge. Tumor-specific cytotoxicity, cytokines, and markers for T cell activation, i.e., CD25 and CD69, were determined 8 days after the boost with huKS1/4-IL-2 fusion protein. Animals were sacrificed 28 days after challenge and analyzed for macroscopic metastases and minimal residual disease by microscopic analysis.
Flow cytometry analysis
Two-color flow cytometric analyses were performed with single-cell suspensions prepared from lymphatic tissues. Anti-CD69 (clone H1.2F3) and anti-CD25 (clone 7D4) were used in PE-conjugated form in combination with FITC-conjugated anti-mouse mAb CD3e (clone 145-2c11). FITC-rat IgM (R4-22) and hamster IgG anti-trinitropherol (G235–2356) were used as Ig isotype controls. Lymphocytes were incubated for 1 h at 4°C with FITC- and PE-labeled Abs and washed and analyzed immediately with a Becton Dickinson FACScan. A total of 10,000 labeled cells per sample were analyzed. All labeled Abs were purchased from PharMingen.
Statistical analyses
The statistical significance of differential findings between experimental groups was determined by the Student’s t test. The nonparametric Wilcoxon rank sum test was chosen when the data for life span and metastatic scores were not amenable for parametric tests. Findings were regarded as significant if two-tailed p values were <0.05.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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We previously demonstrated that an huKS1/4-IL-2 fusion protein
specifically induces a CD8+ T cell-mediated
immune response in syngeneic BALB/c mice capable of completely
eradicating established pulmonary metastases of CT26-KSA colon
carcinoma (8, 10). We initially examined the cellular
mechanisms involved in these events with emphasis on the frequency of
pCTL and subsequent priming of CD8+ T effector
cells. Determinations of the frequency of pCTL in splenocytes of BALB/c
mice with established pulmonary metastases of CT26-KSA colon carcinoma
using a limiting dilution assay at different time points during and
after the huKS1/4-IL-2 fusion protein treatment indicated a 10-fold
increase compared with mice treated with PBS (Fig. 1
A). The increase in pCTL
frequency reached a maximum on day 12 after tumor cell challenge,
corresponding to 1 day after completion of fusion protein treatment
during 7 consecutive days, initiated 4 days after tumor cell
inoculation (Fig. 1B). In contrast, pCTL frequency in
splenocytes obtained from PBS-treated mice remained at background
levels during the 4-wk period examined (Fig. 1B). Although
the frequency of pCTL decreased somewhat from the maximum observed on
day 12, it still remained substantially above the level observed in
control animals even 26 days after tumor cell inoculation (Fig. 1B). The marked increase in the frequency of pCTL observed
correlated with a distinct increase in cytolytic activity of the
splenocytes against CT26-KSA tumor cell targets (Fig. 1C).
Concordantly, the percent cytolysis achieved also reached a maximum on
day 12 post-tumor cell inoculation (Fig. 1D). The
splenocytes obtained from control animals treated with PBS showed only
background levels (<10%) of cytolytic activity against CT26-KSA tumor
target cells (Fig. 1, C and D).
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In an attempt to further characterize priming, proliferation, and
maintenance of CD8+ T cells induced by the IL-2
fusion protein, an RPA was found sensitive in detecting and
quantitating cytokine genes at the RNA level. Analysis of total RNA
extracted from splenocytes obtained from BALB/c mice 12 days after
CT26-KSA tumor cell inoculation and quantitation with a multiprobe RPA
of cytokine gene expression revealed markedly increased levels of genes
encoding Th1 cytokines IL-2, IFN-, TNF-, LT-ß, and GM-CSF only
in mice treated with the IL-2 fusion protein. This was evident from
quantitation of autoradiographic intensity compared with that of the
constitutively expressed L32 control indicating a time-dependent
increase over a period of 48 h (Fig. 2). Although expression of the gene
encoding the Th2 cytokine IL-6 was also induced, genes encoding for the
typical Th2 cytokines IL-4 and IL-10, also quantifiable by this assay,
were not detected (data not shown). By comparison, the expression
levels of genes encoding the cytokines shown in Fig. 2 were
considerably less in RNA extracted from splenocytes of mice treated
with PBS, suggesting that the priming of CD8+ T
cells induced by huKS1/4-IL-2 treatment specifically induces a marked
increase in the expression of genes primarily encoding Th1
cytokines.
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During examination of the mechanism(s) involved in the complete
rejection of a secondary tumor cell challenge in mice cured of
pulmonary metastases by huKS1/4-IL-2 fusion protein, we observed a
14-fold increase in the frequency of pCTL over that detected in
splenocytes from naive mice by using the limiting dilution assay (Fig. 3B). However, in this case,
the cured mice received a secondary tumor cell challenge 6 wk after the
initial tumor cell inoculation. The animals then received a boost 4
days thereafter with two noncurative doses (5 µg each) of
huKS1/4-IL-2 fusion protein on days 4 and 6 after tumor cell challenge.
Importantly, this boost induced not only the marked increase in pCTL
frequency, but also correlated with a decisive increase in cytolytic
activity of splenocytes of these treated mice against CT26-KSA tumor
cell targets (Fig. 3A). In contrast, neither an increase in
pCTL frequency nor in cytolytic activity was observed when splenocytes
were obtained from mice boosted only with PBS (Fig. 3, A and
B).
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To determine the fate of
CD8+ T effector cells in the absence of tumor Ag
and naive T cells, we adoptively transferred purified
CD8+ T cells from tumor-bearing BALB/c mice
treated with huKS1/4-IL-2 fusion protein to syngeneic SCID mice
(n = 4). In this case, CD8+ T
cells were purified from splenocytes obtained from these mice 12 days
after initial tumor cell inoculation and 1 day after completion of the
7-day treatment with huKS1/4-IL-2 fusion protein. The
CD8+ T cells were parked for 5 wk in the SCID
mice, and then these animals’ splenocytes were subjected to FACS
analysis for the presence of CD8+ T cells. As
indicated in Fig. 4, there was a
continuous decrease in the number of CD8+ T
cells, until after 5 wk <5% of CD8+ T cells
were detectable, suggesting that the majority of these T effector cells
apoptosed.
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A. In these experiments, the
SCID mice were challenged with CT26-KSA tumor cells at the 6-wk time
point and then received 4 days later boosts with either PBS, a mixture
of huKS1/4 with rhIL-2, or the huKS1/4-IL-2 fusion protein. The
putative T memory cells remaining in these mice after 6 wk recognized
the tumor Ag presented, as shown by markedly increased generation of
CD8+ T cells in mice treated with the IL-2 fusion
protein, whereas the controls revealed only a minor increase in
CD8+ T cells (Fig. 5A). Importantly,
when the CD8+ T cells from each of these
experiments were examined for their ability to lyse CT26-KSA target
cells, only the CD8+ T cells obtained from mice
boosted with the huKS1/4-IL-2 fusion protein were primed and
effectively killed the tumor cells in vitro (Fig. 5B).
Further evidence for strong priming of these same
CD8+ T cells is documented by their strong
expression of CD25, a marker for IL-2 receptors (Fig. 5C)
and CD69, an early activation marker of T cells (Fig. 5D).
The effective priming of these CD8+ T effector
cells was further substantiated by their release of Th1 cytokines
IFN-, IL-2, and GM-CSF measured by a sandwich ELISA 12 days after
tumor cell challenge and boosts with the huKS1/4-IL-2 fusion protein.
In contrast, release of these cytokines was markedly less from
CD8+ T cells of mice boosted only with PBS (Fig. 6).
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We determined whether CD8+ T cells
adoptively transferred from immune mice to syngeneic SCID mice and
parked there for 6 wk could maintain effective and long-lived memory in
the absence of tumor Ag and naive T cells. To this end, such SCID mice
(n = 4) were challenged with CT26-KSA tumor cells and
boosted 4 days thereafter with two noncurative doses (5 µg each) of
either huKS1/4-IL-2 fusion protein or a nonspecific ch14.18-IL-2 fusion
protein. The data depicted in Table I
indicate that putative CD8+ T memory cells that
had been parked in SCID mice effectively recognized a secondary
challenge with CT26-KSA cells only when boosted with noncurative doses
of huKS1/4-IL-2. In fact, these cells were able to completely reject
this challenge and prevent the establishment of pulmonary metastases
(Table I). This effect was specific, since it did not occur when boosts
were done with the nonspecific fusion protein, ch14.18-IL-2. Also,
boosts with noncurative doses of huKS1/4-IL-2 proved ineffective unless
the adoptively transferred CD8+ T cells were
obtained from BALB/c mice that had been successfully treated with
huKS1/4-IL-2 fusion protein (Table I).
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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We demonstrated that this treatment of tumor-bearing BALB/c mice
resulted in a 10- to 14-fold increase in frequency of pCTL, followed by
the induction of genes encoding Th1 cytokines (IFN-, IL-2, TNF-,
LT-ß, GM-CSF) and the generation of tumor-specific
CD8+ T effector cells, some of which
differentiated into long-lived T memory cells. The frequency of pCTL
correlated with enhanced immune protection against tumor cell
challenge, and long-lived T memory cells could be maintained in
syngeneic SCID mice in the absence of tumor Ag. This was demonstrated
when a tumor cell challenge and subsequent boost of these mice with two
noncurative doses of IL-2 fusion protein resulted in effective
reactivation of CD8+ T effector cells with
concurrent release of Th1 cytokines IFN-, IL-2, and GM-CSF, followed
by complete rejection of the tumor cell challenge and prevention of
pulmonary metastases.
The increase in pCTL frequency induced specifically by the huKS1/4-IL-2
fusion protein correlated with increased tumor-protective immunity in
our tumor model. A critical assumption in the determination of pCTL
frequency by the linear regression analyses of data obtained in the
limiting dilution assay is a good fit of experimental data with the
Poisson distribution. This is true for our data with a mean Y-intercept
(a) equaling 96 and the mean coefficient of determination
(r2) being 0.98. However, the efficiency of
detection of these CTL precursors cannot be assessed with certainty,
and the frequency of pCTL reported should be considered as minimal
estimates (23). Importantly, the increase in pCTL
frequency correlated well with an increase in cytolytic activity of
mouse splenocytes against CT26-KSA tumor target cells, indicating
effective priming of T effector cells. There was also direct evidence
for the involvement of Th1 CTL by the concurrent induction of genes
encoding such Th1 cytokines as IFN-, IL-2, TNF-, LTß, and
GM-CSF, as well as the Th2 cytokine IL-6. Since this up-regulation of
Th1 cytokine genes occurred only in IL-2 fusion protein-treated mice,
it appears that these cytokines were newly synthesized as a consequence
of this therapy and were not released from pre-existing stores. In this
regard, it was recently reported that proliferation and differentiation
of CD8+ T cells to generate cytotoxic lymphocyte
responses is enhanced by inflammatory cytokines, such as IFN-,
produced by macrophages or dendritic cells, and that they can provide a
third signal for activation of naive CD8+ T cells
(24). Although there is no direct evidence that this type
of activation occurs in our tumor model, it cannot be ruled
out.
It is of considerable interest that the generation of increased pCTL frequency could again be achieved by huKS1/4-IL-2 fusion protein 6 wk after the initial tumor cell inoculation, following a secondary challenge with CT26-KSA tumor cells. In this case, only two boosts with noncurative doses of the IL-2 fusion protein were required to achieve a 14-fold increase in frequency of pCTL compared with control mice that did not receive such a boost. Additionally, splenocytes from these BALB/c mice exhibited strong tumor-specific cytolytic activity against CT26-KSA tumor target cells in vitro that was not evident among control animals. These findings, together with our previous observation that under these circumstances secondary tumor cell challenges are completely rejected in 100% of experimental animals, lead to two conclusions. First, increased frequency of pCTL correlates with increased T cell memory. Second, there is an altered and more pronounced responsiveness of T memory cells following a second encounter with the same or related Ag. This "positive" memory occurring after secondary stimulation is known to occur faster, reaches higher peak levels of activity, maintains the response for longer periods, and requires less stringent conditions of costimulation than the primary immune response (25, 26).
The role of persisting Ag in T cell memory and the requirement for
chronic exposure to residual deposits of Ag for maintenance of
CD8+ T cell memory have been the subject of much
discussion and controversy (27, 28, 29). This is in contrast
to CD8+ effector T cells that absolutely require
the presence of Ag consistent with the decrease in adoptively
transferred CD8+ T cells in the absence of Ag, a
fact observed in our system (Fig. 4
). Indeed, there are a number of
reports indicating that memory cells survive poorly on adoptive
transfer unless accompanied by specific Ag (12, 30, 31).
However, the contention that memory cells require constant Ag
stimulation has been challenged by reports demonstrating that
CD8+ memory cells can survive for prolonged
periods after adoptive transfer in the absence of Ag (17, 18, 32). These findings suggest that, at least for
CD8+ cells, some memory T cells do not require
continuous stimulation with Ag for survival. To define the conditions
for maintaining long-lived memory T cells in our tumor model,
CD8+ T cells from immune BALB/c mice were
adoptively transferred to syngeneic SCID mice 12 days after tumor cell
inoculation and parked there for 6 wk. In these animals, which lack
mature T and B lymphocytes, the transferred CD8+
T cells died by apoptosis after 5 wk, when <5% were detectable by
FACS analysis. Apparently, CD8+ T cell memory was
maintained under these circumstances, since a secondary challenge with
CT26-KSA tumor cells after 6 wk, followed by a boost with two small,
noncurative doses of huKS1/4-IL-2 fusion protein, induced the
differentiation of these resting CD8+ T memory
cells into CD8+ effector T cells. This contention
is supported by four lines of evidence. First, there was a pronounced
increase of >40% in CD8+ T cells that apoptosed
to <5% of CD8+ T cells that had been adoptively
transferred to SCID mice and maintained there for 6 wk. Second, these
were primed CD8+ effector T cells that
specifically lysed CT26-KSA target cells in vitro and strongly
expressed markers for IL-2 receptors and early T cell activation
indicated by CD25 and CD69, respectively. Third, these
CD8+ T cells were effectively primed, as they
secreted Th1 cytokines IFN-, IL-2, and GM-CSF. Fourth, and most
importantly, the CD8+ T effector cells exhibited
tumor-protective immunity, as they completely rejected the secondary
tumor cell challenge to SCID mice and prevented the formation of
pulmonary metastases.
Taken together, these data strongly suggest that T cell memory was maintained in SCID mice in the absence of Ag and that tumor cell challenge and boost with huKS1/4-IL-2 fusion protein induced differentiation of resting CD8+ T memory cells into tumor-specific CD8+ T effector cells that rejected the tumor cell challenge. The data obtained in our mouse tumor model suggest that Ab-IL-2 fusion proteins could be of future benefit for treatment in an adjuvant setting of colon cancer patients with minimal residual disease.
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Acknowledgments |
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Footnotes |
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2 Address correspondence and reprint requests to Dr. Ralph A. Reisfeld, The Scripps Research Institute, 10550 North Torrey Pines Road, IMM13, La Jolla, CA 92037. E-mail address:
3 Abbreviations used in this paper: pCTL, precursor CTL; KSA, KS Ag; rhIL-2, recombinant human IL-2; RPA, RNase protection assay; LT, lymphotoxin.
Received for publication April 26, 1999. Accepted for publication July 19, 1999.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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 S. A. Holden, Y. Lan, A. M. Pardo, J. S. Wesolowski, and S. D. Gillies Augmentation of Antitumor Activity of an Antibody-Interleukin 2 Immunocytokine with Chemotherapeutic Agents Clin. Cancer Res., September 1, 2001; 7(9): 2862 - 2869. [Abstract] [Full Text] [PDF]
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A. G. Niethammer, R. Xiang, J. M. Ruehlmann, H. N. Lode, C. S. Dolman, S. D. Gillies, and R. A. Reisfeld Targeted Interleukin 2 Therapy Enhances Protective Immunity Induced by an Autologous Oral DNA Vaccine against Murine Melanoma Cancer Res., August 1, 2001; 61(16): 6178 - 6184. [Abstract] [Full Text] [PDF]
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P. M. J. McLaughlin, M. C. Harmsen, W. H. A. Dokter, B.-J. Kroesen, H. van der Molen, M. G. L. Brinker, H. Hollema, M. H. J. Ruiters, C. H. C. M. Buys, and L. F. M. H. de Leij The Epithelial Glycoprotein 2 (EGP-2) Promoter-driven Epithelial-specific Expression of EGP-2 in Transgenic Mice: A New Model to Study Carcinoma-directed Immunotherapy Cancer Res., May 1, 2001; 61(10): 4105 - 4111. [Abstract] [Full Text]
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 C.-C. Ku, J. Kappler, and P. Marrack The Growth of the Very Large CD8+ T Cell Clones in Older Mice Is Controlled by Cytokines J. Immunol., February 15, 2001; 166(4): 2186 - 2193. [Abstract] [Full Text] [PDF]
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M. Imboden, K. R. Murphy, A. L. Rakhmilevich, Z. C. Neal, R. Xiang, R. A. Reisfeld, S. D. Gillies, and P. M. Sondel The Level of MHC Class I Expression on Murine Adenocarcinoma Can Change the Antitumor Effector Mechanism of Immunocytokine Therapy Cancer Res., February 1, 2001; 61(4): 1500 - 1507. [Abstract] [Full Text]
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) or huKS1/4-IL-2-treated mice (
) by limiting
dilution assay, as described in Materials and Methods.
Data shown are from one of three representative experiments on day 12
after tumor cell inoculation. B, pCTL frequency was
determined over time. Each time point represents the mean ± SD of
at least three separate experiments; huKS1/4-IL-2-treated mice (
) as a function of E:T ratio in a 4-h
51Cr-release assay. D, Temporal assessment
of CTL lytic activity of splenocytes from KS1/4-IL-2-treated mice (
);
huKS1/4 + IL-2 mixture (
); huKS1/4-IL-2 fusion protein (