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The Journal of Immunology, 2006, 176: 7325-7334.
Copyright © 2006 by The American Association of Immunologists

Chronic Immune Therapy Induces a Progressive Increase in Intratumoral T Suppressor Activity and a Concurrent Loss of Tumor-Specific CD8+ T Effectors in her-2/neu Transgenic Mice Bearing Advanced Spontaneous Tumors1

Raji E. Nair, Mehmet O. Kilinc, Stacy A. Jones and Nejat K. Egilmez2

James Graham Brown Cancer Center and Department of Microbiology and Immunology, School of Medicine, University of Louisville, Louisville, KY 40202


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
A single intratumoral injection of IL-12 and GM-CSF-encapsulated microspheres induces the complete regression of advanced spontaneous tumors in her-2/neu transgenic mice. However, tumor regression in this model is transient and long-term cure is not achieved due to recurrence. Posttherapy molecular analysis of immune activation/suppression markers within the tumor microenvironment demonstrated a dramatic up-regulation of IFN-{gamma} and a concomitant down-regulation of Forkhead/winged-helix protein 3 (Foxp3), TGFbeta, and IL-10 expression. Therapy-induced reversion of immune suppression was transient since all three markers of suppression recovered rapidly and surpassed pretherapy levels by day 7 after treatment, resulting in tumor resurgence. Repeated treatment enhanced short-term tumor regression, but did not augment long-term survival. Serial long-term analysis demonstrated that although chronic stimulation enhanced the IFN-{gamma} response, this was countered by a parallel increase in Foxp3, TGFbeta, and IL-10 expression. Analysis of tumor-infiltrating T lymphocyte populations showed that the expression of Foxp3 and IL-10 was associated with CD4+CD25+ T cells. Repeated treatment resulted in a progressive increase in tumor-infiltrating CD4+CD25+Foxp3+ T suppressor cells establishing their role in long-term neutralization of antitumor activity. Analysis of tumor-infiltrating CD8+ T cells demonstrated that although treatment enhanced IFN-{gamma} production, antitumor cytotoxicity was diminished. Monitoring of CD8+ T cells that specifically recognized a dominant MHC class I her-2/neu peptide showed a dramatic increase in tetramer-specific CD8+ T cells after the first treatment; however, continuous therapy resulted in the loss of this population. These results demonstrate that both enhanced suppressor activity and deletion of tumor-specific T cells are responsible for the progressive loss of efficacy that is associated with chronic immune therapy.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
Tumor vaccines can successfully promote the development of tumor-specific cytotoxic T cell responses in both murine tumor models and cancer patients (1, 2). In contrast, induction of antitumor T cells rarely results in clinical tumor regression (3, 4). Postvaccine antitumor T cell immunity is routinely monitored by quantification of tumor-specific T cells in peripheral blood (5). However, peripheral T cell activity is a poor correlate for intratumoral T effector cell function (2, 4, 6), because tumors can actively subvert immune responses by producing immune inhibitory molecules and by attracting and retaining T suppressor cells (7). Therefore, combinatorial approaches that are designed to concurrently prime tumor-specific T cell responses and to neutralize the immune suppressive elements in the tumor microenvironment are more likely to achieve effective tumor regression.

The cytokine milieu of the tumor microenvironment is critical to the outcome of the interaction between the tumor and the immune system (7, 8). Modulation of the tumor microenvironment by local delivery of proinflammatory cytokines can overcome tumor immune suppression and induce both local and systemic antitumor regression (8). To this end, we demonstrated that a single intratumoral injection of IL-12 and GM-CSF-encapsulated biodegradable, sustained-release microspheres can achieve the complete regression of established primary tumors, induce long-term systemic antitumor T cell immunity, and eradicate established metastatic disease in a transplantable murine lung tumor model (9, 10). More recently, we evaluated (11) the curative potential of this in situ vaccination approach in her-2/neu transgenic mice, who spontaneously develop mammary tumors. These studies demonstrated that in situ delivery of IL-12 and GM-CSF achieved complete regression of advanced spontaneous mammary tumors and promoted the development of long-term, protective T and B cell responses. In contrast to the results obtained in the transplantable tumor model, tumor eradication was found to be transient in this model because most lesions eventually recurred. Repeated treatment of recurring tumors improved therapeutic efficacy in the short term; however, long-term survival was not enhanced beyond what was achieved with a single treatment due to recurrence. Preliminary analysis of tumors in mice receiving repeated treatment demonstrated a progressive decline in the intensity of posttherapy T cell activity within the tumor microenvironment (11).

Recently, it was reported (12) that repeated vaccination promotes a progressive quantitative increase in the frequency of peripheral antitumor T cells, but that this increase does not result in enhanced tumor regression in patients. Functional integrity of vaccine-induced, tumor-infiltrating T cells was not monitored in this study. Because chronic stimulation failed to maintain intratumoral T cell activity in our studies, we hypothesized that the observed lack of correlation between enhanced peripheral T effector activity and tumor regression could be due to a concurrent augmentation of T suppressor cell activity in the tumor microenvironment. To this end, quantitative monitoring of T effector cell activity and the accompanying T suppressor cell responses in tumors has not been performed during chronic therapy. To determine whether repeated treatment of advanced spontaneous mammary tumors in her-2/neu transgenic mice resulted in such a response-counterresponse process, we monitored the quantitative and qualitative changes in immune activation and suppression markers in posttherapy tumors. The results establish that intratumoral delivery of IL-12 and GM-CSF promotes a dramatic reversal of immune suppression in the tumor microenvironment but that this reversion is transient because intratumoral T suppressor cell activity recovers rapidly. Importantly, our data demonstrate that whereas repeated treatment resurrects antitumor immune activity, reactivation is countered by a concurrent and progressive increase in the intensity of T suppressor cell infiltration into tumors, resulting in reduced T effector cytotoxicity and eventual deletion of tumor-specific CTL.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 Disclosures
 References
 
Mice

Five breeder pairs of FVB/N or FVB/N-TgN MMTVneu202Mul (FVBneuN) at 6–8 wk of age were purchased from The Jackson Laboratory. Mice were bred and female progeny were maintained in microisolation cages (Laboratory Products) in the barrier unit of the University of Louisville’s Laboratory Animal Facility. All other strains were purchased from The Jackson Laboratory. Tumor development was monitored by palpation once a week after mice reached 4 mo of age. All studies were approved by the Institutional Animal Care and Use Committee of the University of Louisville.

Cytokines and microspheres

Recombinant murine IL-12 (2.7 x 106 U/mg) was a gift from Wyeth-Averst Pharmaceuticals. Recombinant murine GM-CSF (5 x 106 U/mg) was purchased from PeproTech. Preparation of cytokine-encapsulated biodegradable polymer microspheres was described in detail previously (11).

Microsphere treatments, monitoring of tumor growth, and survival

Mice were monitored for tumor development by palpation twice a week. All mice developed tumors between 175 and 330 days of age. Treatment was administered with a single intratumoral injection of microspheres when tumors reached ~100–200 mm3 in size (tumor volume was determined according to the formula A x B2/2, where A is the longest and B is the shorter perpendicular dimension of the tumor). Experimental groups received IL-12 plus GM-CSF-encapsulated microspheres (bioactivity equivalent of 2 µg cytokine) suspended in 150 µl of hydration buffer (1% hydroxypropylmethylcellulose (Dow) and 1% Pluronic F-127 (Sigma-Aldrich) in PBS, pH 7.2). Control mice received blank microspheres in 150 µl of hydration buffer. Mice were sacrificed when the largest diameter of the tumor reached 15 mm.

Quantitative real-time PCR analysis

Fine needle aspirates were obtained by aspirating four quadrants of each tumor with a 233/4-gauge needle attached to a 1.0-ml syringe. Tissue samples were discharged into TRIzol (Invitrogen Life Technologies); total mRNA was isolated and was reverse-transcribed with TaqMan Reverse Transcription reagents (Applied Biosystems). IFN-{gamma}, IL-10, Forkhead/winged-helix protein 3 (Foxp3)3, TGF-beta, indoleamine 2,3 dioxygenase (IDO), and GAPDH mRNA levels were quantified by real-time RT-PCR amplification using the Mx3000PTM Real-Time PCR System (Stratagene) as recommended by the manufacturer. Briefly, target transcripts were amplified in a 25-µl reaction mixture containing 12.5 µl of SYBR Green PCR Master Mix (Applied Biosystems), 100 ng of cDNA template, and selected primers (200 nM) using the recommended cycling conditions (denaturation at 95°C for 10 min followed by 40 cycles of 95°C for 15 s and 60°C for 1 min). The primer sequences, designed with Primer Express software (Applied Biosystems), were as follows: Foxp3, 5'-TCCCACGCTCGGGTACAC-3' (forward) and 5'-TTGCCAGCAGTGGGTAGGAT-3' (reverse); IFN-{gamma}, 5'- GCACAGTCATTGAAAGC-3' (forward) and 5'- TGCCAGTTCCTCCAGATA-3' (reverse); IL-10, 5'-CCTGGTAGAAGTGATGCCCC-3' (forward) and 5'-TCCTTGATTTCTGGGCCATG-3' (reverse); GAPDH, 5'-TCCTTGATTTCTGGGCCATG-3' (forward) and 5'-TCTTCTGGGTGGCAGTGATG-3' (reverse); TGF-beta, 5'-CGCTTGCAAAACCCCAAA-3' (forward) and 5'-TTTCCATTTAAGGATCTGA TACAGTTCA-3' (reverse); and IDO, 5'-CAGGCCAGAGCAGCATCTTC-3' (forward) and 5'-GCCAGCCTCGTGTTTTATTCC-3' (reverse). Relative quantification of mRNA expression was calculated by the comparative cycle threshold (Ct) method (13). The relative target quantity, normalized to an endogenous control (GAPDH) and relative to the day 0 calibrator, was expressed as 2{Delta}{Delta}Ct (fold), where {Delta}Ct = Ct of the target gene (IFN-{gamma}, Foxp3, IL-10, TGF-beta) – Ct of endogenous control gene (GAPDH), and {Delta}{Delta}Ct = {Delta}Ct of samples for target gene – {Delta}Ct of the 0-day calibrator for the target gene.

Preparation of single-cell suspensions from tumors

Tumors were disaggregated by a modification of the enzyme digestion technique originally described by Russell et al. (14). Briefly, tumors were excised and minced with sterile scissors and a surgical blade into ~1–2 mm3 pieces. The minced tumor fragments were then digested in DMEM/F12 containing 10% FCS and 0.05 mg/ml collagenase D (Roche Molecular Systems), 0.02 mg/ml hyaluronidase type V (Sigma-Aldrich), and 0.01 mg/ml DNase I (Sigma-Aldrich), in PBS plus 0.5% BSA at 37°C in a rotating platform for 1 h. The supernatant was filtered through 70-µm nylon mesh (Falcon 2340; BD Biosciences) to remove clumps and washed immediately in cold DMEM/F12. The cell suspension containing 5–10 x 107 viable cells in 5 ml of DMEM/F12 was layered over 3 ml of 20% Ficoll (Amersham Biosciences) solution in a 15-ml conical bottom centrifuge tube and centrifuged for 20 min at 1350 x g. Live cells (tumor cells and tumor-infiltrating leukocytes (TIL)) were collected from the interface and analyzed by flow cytometry.

Flow cytometry

Cells were stained using standard techniques (15) and analyzed on a four-color BD FACSCalibur flow cytometer (BD Biosciences). The following mAbs were used: FITC-conjugated anti-mouse CD4 (GK1.5), FITC- conjugated anti-mouse CD8 (53-6.7), and allophycocyanin-conjugated anti-mouse CD25 (PC61). Background levels were determined with isotype-matched control Abs. All Abs were purchased from BD Pharmingen.

Intracellular cytokine staining

Intracellular staining for IFN-{gamma} and IL-10 was performed using the BD Pharmingen kit for detection of IFN-{gamma} and IL-10 as recommended by the manufacturer (BD Pharmingen). Briefly, 1 x 106 cells were incubated in CTL medium (RPMI 1640 plus 10% FBS, 0.5% L-glutamine, 10 µg/ml penicillin/streptomycin, and 50 µM 2-ME, pH 7.1) containing PMA (50 ng/ml) and ionomycin (500 ng/ml) in the presence of GolgiStop. Cells were washed in FACS buffer (BD Pharmingen), stained with FITC-conjugated anti-mouse CD8 (53-6.7), fixed, permeabilized, and stained with PE-conjugated anti-mouse IFN-{gamma} (XMG1.2). For detection of intracellular IL-10, cells were stained with FITC-conjugated anti-mouse CD4 (GK1.5) and allophycocyanin-conjugated anti-mouse CD25 (PC61), fixed, permeabilized, and stained with PE-conjugated anti-mouse IL-10 (JES5-16E3). Intracellular staining for Foxp3 was performed using a Foxp3 staining kit (eBioscience). Cells were stained with FITC-conjugated anti-mouse CD4(GK1.5) and allophycocyanin-conjugated anti-CD25 (PC61), fixed, permeabilized, and stained with PE-conjugated anti-mouse Foxp3 (FJK-16s; eBioscience). All Abs were from BD Pharmingen unless indicated otherwise.

IFN-{gamma} ELISPOT assay

Assays were performed using the Mouse IFN-{gamma} ELISPOT plus kit (Mabtech). Briefly, 96-well ELIIP10SSP polyvinylidene fluoride plates (Millipore) were coated at 4°C overnight with 3 µg/ml capture Ab (anti-IFN-{gamma} Ab AN-18; Mabtech). The plates were then washed and blocked with DMEM/F12 with 10% FCS for 1 h at 37°C. TIL or tumor-draining lymph nodes (TDLN) cells (2 x 105/well) were cultured in 100 µl of culture medium either with anti-mouse CD3 (1µg/ml; BD Pharmingen) plus IL-2 (5 ng/ml) or RNEU420–429 peptide PDSLRDLSVF (16) for 48 h at 37°C and 5% CO2 in duplicate wells with 1/2 serial dilutions. After culture, the plates were washed and incubated first with 1 µg/ml biotinylated anti-IFN-{gamma} Ab (Mabtech), then with 1/1000 streptavidin-alkaline phosphatase (Mabtech), and finally with 5-bromo-4-chloro-3-indolyl phosphate/NBT; Mabtech). The plates were developed at room temperature for 20–30 min until visible spots appeared. The reaction was then stopped by washing extensively in tap water. The plates were air-dried, and the spots were counted with a dissecting microscope. The frequency of cytokine-producing cells was expressed as the difference between the mean number of spots after stimulation and the mean background without stimulation. The background was 9.4 ± 2.3 spots/well.

Cytotoxicity assays

Mice were euthanized by CO2 inhalation, TDLN were removed and mechanically disaggregated through a 70-µm cell strainer. The cell suspension was then enriched for CD8+ T cells using the SpinSep-negative selection separation system according to the manufacturer’s instructions (StemCell Technologies). The purity of the CD8+ T effector cell preparation was >95% as determined by flow cytometry. Cell-mediated cytotoxicity was measured by a dye exclusion assay (17, 18). Target cells were prepared and pooled from established tumors of six to eight mice as described previously (11). After washing once in PBS, tumor cells were stained with PKH26 using the PKH26-GL dye kit (Sigma-Aldrich) as recommended by the manufacturer. PKH26-labeled target cells were then suspended at 2–4 x 105 cells/ml in DMEM/F12 and 100-µl aliquots were added to FACS tubes for the CTL assay. CD8+ T effectors (100 µl) at various concentrations were mixed with target cells, tubes were centrifuged at 200 x g for 2 min, and incubated at 37°C for 48 h. Following incubation, 1 µl of 1 mg/ml stock solution of propidium iodide was added to each tube and the tubes were incubated for 15 min at 37°C before analysis by flow cytometry. Five thousand PKH26-labeled target cells were acquired per sample. The extent of cytotoxicity was determined by quantification of dead cells labeled with both PKH-26 plus propidium iodide and live cells labeled with PKH-26. Cytotoxicity was reported as percentage of cell death within the PKH-26 plus targets ((dead-labeled targets/dead-labeled targets plus live-labeled targets) multiplied by 100). Percentage target cell death was corrected for spontaneous background death by subtracting the percentage of dead cells in control samples (PKH-26-labeled targets alone) from the percentage of dead cells within the test samples.

Tetramer analysis

Recombinant allophycocyanin-conjugated H-2Dq tetramers bound to neu-specific peptide PDSLRDLSVF were produced by the MHC Tetramer Core Facility at the National Institute of Allergy and Infectious Diseases (Bethesda, MD). The TIL (prepared as single-cell suspensions from tumors as described above) and TDLN cells were stained with H-2Dq tetramer for 30 min on ice and surface stained with FITC-conjugated anti-mouse CD8 (53-6.7). Cells were then fixed and permeabilized with a Cytofix/Cytoperm kit (BD Pharmingen), followed by intracellular staining for IFN-{gamma} as described above.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 Disclosures
 References
 
A single injection of IL-12 plus GM-CSF microspheres results in a dramatic reversal of intratumoral immune suppression and promotes enhanced survival

To determine how repeated treatment with IL-12 and GM-CSF-loaded microspheres affected tumor-infiltrating T lymphocyte activity, we monitored the expression of intratumoral T cell activation (IFN-{gamma}) and suppression (Foxp3, TGFbeta, and IL-10) markers by quantitative real-time PCR. Initially, we analyzed the effect of therapy on the selected markers in short-term studies to demonstrate that treatment altered their expression profiles, thus validating the rationale for this approach. Mice bearing spontaneous advanced (100–200 mm3) mammary tumors were treated either with a single intratumoral injection of IL-12 plus GM-CSF-encapsulated microspheres or control (blank) microspheres. Tissue samples were obtained from tumors by fine needle aspiration (FNA) before therapy and at different time points following treatment (19), and total RNA was analyzed for IFN-{gamma}, Foxp3, TGFbeta, and IL-10 transcript levels by real-time PCR. The results are shown in Fig. 1. These data demonstrate that treatment promoted a rapid and dramatic increase in intratumoral IFN-{gamma} transcript levels as early as 6 h posttherapy, which continued to increase until day 3 (Fig. 1A). More importantly, the dramatic up-regulation of IFN-{gamma} during the first 3 days was accompanied by a concomitant decrease (up to 4-fold) in markers of immune suppression, i.e., Foxp3, TGFbeta, and IL-10 (Fig. 1, B–D), demonstrating an effective reversal of the immune suppressive characteristics of the tumor microenvironment. In contrast, control-treated mice neither showed a significant increase in IFN-{gamma} (Fig. 1A, inset) nor a decrease in any of the suppression markers (Fig. 1, B–D, insets). Treatment-induced reversal of immune suppression resulted in improved long-term survival in treated mice as compared with control-treated animals (Fig. 1E).


Figure 1
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  		FIGURE 1. Effect of IL-12 + GM-CSF microsphere treatment on intratumoral IFN-{gamma}, Foxp3, TGFbeta, and IL-10 expression. FNA samples were obtained from tumors before treatment and at indicated times after treatment. Total RNA was analyzed for the expression of selected markers by real-time PCR as described in Materials and Methods. The fold change is relative to pretreatment levels of each transcript (1-fold). A–D, Samples were obtained from six to eight mice per time point. Error bars, SD. Insets, The results from mice treated with control (blank) microspheres. E, Survival of mice after a single treatment. Control mice received an injection of blank microspheres. The difference between the survival of control and IL-12 plus GM-CSF groups was highly significant as determined by log-rank analysis (p = 0.0007, n = 14 and n = 12 for control and IL-12 plus GM-CSF, respectively).

 
Treatment-induced reversal of intratumoral immune suppression is transient and repeated treatment results in progressively higher rebound levels of Foxp3, TGFbeta, and IL-10 in tumors

The above results demonstrated that IL-12 plus GM-CSF microsphere treatment resulted in a dramatic conversion of the immune-suppressive tumor milieu to one that is immunologically active, at least in the short term. To determine whether this reversion persisted, posttherapy intratumoral IFN-{gamma}, Foxp3, TGFbeta, and IL-10 levels were monitored further, initially up to 20 days after the first treatment, and then for up to 7 wk during repeated therapy. The long-term real-time PCR data for the four selected markers are shown in Fig. 2. These results again demonstrate that treatment with IL-12 plus GM-CSF microspheres reversed the immune suppressive character of the tumor microenvironment as indicated by the >30-fold increase in intratumoral levels of IFN-{gamma} and a 2- to >10-fold decrease in the levels of Foxp3, TGFbeta, and IL-10 within 2–3 days after treatment. This reversal, however, was transient and transcript levels of all three markers of suppression recovered rapidly. This recovery proceeded despite the continued presence of high quantities of IFN-{gamma} and in fact surpassed pretreatment levels by 10- to 20-fold as early as day 7. By day 20 posttreatment, IFN-{gamma} expression had declined; however, Foxp3, TGFbeta, and IL-10 transcript levels remained high and, in the case of IL-10 and TGFbeta, continued to increase, reaching >200-fold above pretherapy levels (Fig. 2). These data establish that treatment-induced reversal of tumor immune suppression was brief and was followed by a rapid recovery of suppression markers to levels that exceeded pretherapy intensity.


Figure 2
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  		FIGURE 2. Effect of repeated IL-12 plus GM-CSF microsphere treatment on long-term IFN-{gamma}, Foxp3, TGFbeta, and IL-10 expression in tumors. FNA samples were obtained from tumors before treatment (day 0) and on indicated days after treatment. Total RNA from each sample was analyzed for the expression of selected markers by real-time PCR as described in Materials and Methods. The fold change is relative to day 0 pretreatment levels of each transcript. Values are averages of samples from three to eight mice per time point. FNA samples could not be obtained from one mouse after day 7 due to complete tumor regression and from three other mice at different time points due to transient tumor regression at the time. After day 35, three mice had to be sacrificed due to tumor growth before FNA samples could be procured. Error bars, SD. Arrows indicate time of treatment (days 0, 21, and 42).

 
To determine whether the T cell activity within the tumor microenvironment could be resurrected, tumors were injected again on days 21 and 42 after the first treatment. Upon administration of the second treatment on day 21, a significant increase in IFN-{gamma} and a concomitant decline in Foxp3, TGFbeta, and IL-10 levels were achieved again (Fig. 2). In fact, the second treatment induced much higher levels of IFN-{gamma} than the first treatment (>190-fold increase over pretherapy levels on day 24 vs 30-fold on day 3). Consistent with this increase, the reversal of suppression was highly effective, resulting in >200-, 70-, and 500-fold decreases in Foxp3, TGFbeta, and IL-10 transcript levels between days 20 and 24, respectively (Fig. 2, B–D). In contrast, analysis of day 28 samples showed a rapid recovery of the suppressive factors between days 24 and 28 similar to that seen after the first treatment, except that the absolute transcript levels were now significantly higher (cf. days 7 and 28). After the third treatment (on day 42), there was again a dramatic increase in IFN-{gamma} levels (>9000-fold higher than pretherapy levels); however, suppression of Foxp3, TGFbeta, and IL-10 was less effective than before (cf. changes between days 41 and 45). In fact, TGFbeta no longer appeared to respond to treatment. The absolute transcript levels for suppression markers 1 wk after the third treatment (day 49) were higher than the levels reached after the second treatment (>195-, 190-, and 280-fold above pretherapy levels on day 49 vs >85-, 20-, and 65-fold on day 28 for Foxp3, TGFbeta, and IL-10, respectively), demonstrating the progressive nature of regulatory recovery with each treatment. These results establish, in a quantitative manner, that although repeated treatment promoted progressively stronger immune activation in the tumor microenvironment as measured by IFN-{gamma} levels, suppressive mechanisms countered this activation with increasing intensity.

The progressive increase in intratumoral Foxp3 and IL-10 expression that accompanies repeated treatment is associated with enhanced T suppressor cell infiltration

Because Foxp3, TGFbeta, and IL-10 are all expressed by CD4+CD25+ T suppressor cells, we quantitatively monitored CD4+CD25+ T cells in pre- and posttherapy tumors to determine whether repeated treatment resulted in increased T suppressor infiltration. For this, lymphocyte populations were isolated from tumors as well as TDLN before treatment (day 0), 1 wk after the first treatment (day 7), and 1 wk after the third treatment (day 49), and CD4+CD25+ T cells were quantified by flow cytometry. The results are shown in Fig. 3. There were no differences in the percentage of CD4+ T cells in the TDLN obtained from 0-, 7-, and 49-day mice (Fig. 3A). In contrast, the proportion of the CD4+ T cells within the TIL increased progressively between days 0 and 49 (>4-fold; Fig. 3A and Table I). The proportion of the CD4+CD25+ subset within the CD4+ T cell population did not change in the TDLN but expanded in tumors (1.5- to 3.8-fold in different experiments) between days 0 and 49 (Fig. 3B). The overall increase in CD4+CD25+ cells within the TIL population exceeded 12-fold between days 0 and 49 (Table I). The progressive rise in the CD4+CD25+ T cell numbers between days 0 and 49 mirrored the real-time PCR data; therefore, suggesting that the increase in Foxp3, TGFbeta, and IL-10 transcript levels was associated with enhanced T suppressor activity. To confirm that the expression of suppression markers was associated with the CD4+CD25+ T cells found in the tumor, the cells were further analyzed for Foxp3 and IL-10 expression by intracellular cytokine staining. Approximately 3% of intratumoral CD4+CD25+ T cells expressed Foxp3 and ~10% expressed IL-10 on day 0, demonstrating limited T suppressor cell presence in untreated tumors (Fig. 3C). The proportion of intratumoral CD4+CD25+ T cells expressing Foxp3 however, increased with each treatment (up to 43% on day 49), establishing that chronic stimulation resulted in a dramatic enhancement of T suppressor infiltration and/or expansion (Fig. 3C). To this end, quantitative analysis of data from multiple experiments established that the fraction of CD4+CD25+Foxp3+ cells in TIL increased by >100-fold between days 0 and 49 (Table I). A similar increase in IL-10 expression by tumor-infiltrating CD4+CD25+ T cells (from 10 to 26%) was also observed following repeated treatment (Fig. 3C). Collectively, these data establish that the progressive increase in the expression of suppression markers during repeated treatment was the result of enhanced CD4+CD25+ T suppressor cell infiltration into tumors.


Figure 3
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  		FIGURE 3. Effect of treatment on intratumoral T suppressor cell activity. TDLN cells and single-cell suspensions prepared from tumors were analyzed by four-color flow cytometry as described in Materials and Methods. Cells were pooled from three separate mice for each time point. Results are representative of at least two, in some cases three, independent experiments. A, Lymphocytes were gated on and analyzed for CD4 expression. B, CD4+ cells were gated on and analyzed for CD25 expression. C, CD4+CD25+ T cells were gated on and analyzed for Foxp3 and IL-10 expression by intracellular staining. Gray shading, isotype control; red, day 0; blue, day 7; and green, day 49.

 

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  		Table I. Fold increase in Foxp3+ CD4+ CD25+ T-cells in tumors between days 0 and 49a

 
Enhanced T suppressor activity does not affect CD8+ T cell IFN-{gamma} production but impairs cytotoxic function

Previous studies (11) in our laboratory demonstrated that intratumoral IL-12 plus GM-CSF therapy induced a potent antitumor CD8+ cytotoxic T cell response, which was required for tumor eradication. Although the above studies established that enhanced intratumoral Foxp3, IL-10, and TGFbeta expression was associated with increased T suppressor activity, whether this resulted in the inhibition of antitumor CTL function was not determined. The initial finding that repeated treatment augmented intratumoral IFN-{gamma} expression with increasing efficacy did not support this notion. To determine whether T cells were a significant source of IFN-{gamma}, their ability to produce IFN-{gamma} was analyzed by ELISPOT assay. Lymphocytes were isolated from tumors or the TDLN on days 0, 7, and 49, stimulated either with IL-2 and anti-CD3 Ab or a her-2/neu MHC class I peptide, and analyzed for IFN-{gamma} production. The results are shown in Fig. 4. These data demonstrate that the ability of T cells to produce IFN-{gamma} in response to anti-CD3 Ab stimulation did not diminish, but in fact increased with repeated treatment, and are consistent with the findings from the FNA/RT-PCR studies (Fig. 2A). Moreover, pulsing of cells with a her-2/neu MHC class I peptide also resulted in a progressive increase in IFN-{gamma}-secretion (particularly within the TIL), suggesting that tumor-specific CD8+ T cells were responsible for a significant portion of IFN-{gamma}-production (Fig. 4). These data establish that repeated treatment augmented IFN-{gamma}-production by T cells despite increased intratumoral T suppressor activity.


Figure 4
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  		FIGURE 4. ELISPOT analysis of IFN-{gamma} production by T cells during repeated treatment. Cells were isolated from tumors (TIL) and the TDLN and analyzed for IFN-{gamma} production by ELISPOT assay on days 0 (pretreatment), 7 (1 wk after the first treatment), and 49 (1 wk after the third treatment) as described in Materials and Methods. Cells from three mice were pooled and processed in duplicate for each time point. In the TIL study, the differences between all time points were significant (p < 0.048) for the her-2/neu peptide group. In the anti-CD3/IL-2 group, differences between days 0 vs 49 and 7 vs 49 were significant (p < 0.0007). In the TDLN study, the difference between days 0 and 49 was significant in the anti-CD3/IL-2 group (p = 0.012). Error bars, SD.

 
ELISPOT analysis provided strong evidence that increasing T-suppressor activity did not compromise T cell function in general, or intratumoral CD8+ T cell activity in particular. To directly determine whether chronic stimulation enhanced the quantity and/or function of intratumoral CD8+ T cells, we next monitored the CD8+ T cell populations obtained from the TDLN and tumors during repeated treatment. Flow cytometric analysis of CD8+ T cells showed no changes in the percentage of CD8+ T cells on days 7 and 49 compared with day 0 in the TDLN (Fig. 5A). In contrast, the quantity of CD8+ T cells within the TIL population increased by >3-fold between days 0 and 49 (Fig. 5A). To determine whether the increased T suppressor infiltration affected the ability of CD8+ T cells to produce IFN-{gamma}, CD8+ T cells from the TDLN and TIL were analyzed by intracellular cytokine staining. The results demonstrate that treatment induced IFN-{gamma} expression by both TDLN and tumor-infiltrating CD8+ T cells with increasing efficacy after each injection, in agreement with the findings of FNA/RT-PCR and ELISPOT analyses (Fig. 5B). This increase was particularly dramatic within the tumor (~6-fold and ~22-fold increase in the proportion of IFN-{gamma}-positive CD8+ T cells on days 7 and 49, respectively). Collectively, these data demonstrate that treatment-induced T suppressor activity did not diminish the ability of CD8+ T cells to infiltrate the tumors or produce IFN-{gamma} in response to repeated therapy.


Figure 5
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  		FIGURE 5. Effect of repeated treatment on intratumoral CD8+ T cell activity. The ability of CD8+ T cells isolated from the TDLN and tumors (TIL) to produce IFN-{gamma} and mediate cytotoxic killing were evaluated. A, The proportion of CD8+ T cells within the TDLN or TIL populations was quantified during repeated treatment. B, Percent IFN-{gamma}-positive cells within the CD8+ T cell population. CD8+ T cells were gated on and analyzed for IFN-{gamma} expression by intracellular staining. The results shown for A and B are representative of at least two, in some cases three, independent experiments. Gray shading, isotype control; red, day 0; blue, day 7; and green, day 49. C, Cytotoxic killing of her-2/neu-expressing tumors by CD8+ T cells before treatment (day 0), 1 wk after the first treatment (day 7), and 1 wk after the third treatment (day 49). Cells were pooled from the TDLN of three mice per group. Combined data from two experiments are shown. The differences between days 0 and 7 and days 7 and 49 were statistically significant (p = 0.0001 and 0.037, respectively) at the E:T ratio of 10:1. Error bars, SD.

 
A recent study (20) showed that although T suppressor cells do not significantly impair the ability of CTL to produce cytokines in vivo, they effectively inhibit cytotoxic function through a TGFbeta-dependent mechanism. To determine whether this was true in our model, CD8+ T cells were isolated from the TDLN of mice on days 0, 7, and 49 and their ability to mediate tumor killing was tested in vitro (Fig. 5C). These results demonstrate that although the T cells isolated from day 0 mice mediated very little or no killing of tumor targets, a single treatment with IL-12 plus GM-CSF microspheres induced a dramatic increase in the ability of CD8+ T cell populations isolated (on day 7) from the TDLN to lyse tumor cells. In contrast, after three successive treatments, day 49 CD8+ T cells demonstrated a significant reduction in cytotoxic function. These data establish that the progressive increase in the quantity of intratumoral T suppressor cells is associated with a concurrent decrease in the ability of cytotoxic CD8+ T cells to mediate tumor killing.

Loss of CD8+ T cell cytotoxic function is accompanied with deletion of tumor-specific CTLs

The studies above focused on the total CD8+ T cell population but did not investigate their tumor specificity. It has been shown (21) that her-2/neu, which is overexpressed by tumors in this strain represents a bona fide tumor Ag that can be recognized by CTL. More recently (16), a MHC class I epitope was identified for rat her-2/neu. To determine whether IL-12 plus GM-CSF microsphere treatment induced her-2/neu-specific CD8+ T cells and to establish how these cells responded to repeated treatment, tetramer analysis of her-2/neu-specific CD8+ T cells was performed. Treatments were administered on days 0, 21, and 42, cells were isolated from the tumors and the TDLN on days 0 (before therapy), 7, and 49, and tetramer-specific CD8+ T cells were quantified by flow cytometry (Fig. 6). Analysis of tumor-infiltrating CD8+ T cells in untreated tumor-bearing mice and splenic CD8+ T cells of non-tumor-bearing control mice demonstrated identical levels of tetramer-positive cells, suggesting that the mice were tolerant to her-2/neu (Fig. 6, Control). In contrast, a single injection of IL-12 plus GM-CSF microspheres induced a dramatic (>12-fold) increase in the quantity of her-2/neu-specific CD8+ T cells in the tumor (from 3 to 36%) confirming that intratumoral IL-12 plus GM-CSF promotes a potent CTL response that infiltrates tumors successfully (Fig. 6, TIL). A significant 4-fold increase in tetramer-positive cells is also observed in the TDLN on day 7 (Fig. 6, TDLN). More importantly, however, following repeated treatment, a 30-fold reduction in the quantity of tetramer-specific CD8+ T cells is observed in the tumor on day 49 in comparison to day 7 (Fig. 6, TIL). These findings demonstrate that the reduction in the cytotoxic function of the CD8+ T cell component is paralleled with extensive changes in the clonal composition of CD8+ T cell populations in the long term.


Figure 6
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  		FIGURE 6. Induction of her-2/neu-specific cytotoxic T cells by IL-12 plus GM-CSF microsphere treatment. Control, The presence of her-2/neu-specific CD8+ T cells in the spleens of BALB/c, wild-type FVB/NJ, and tumor-bearing untreated FVBneuN mice were evaluated by a tetramer-binding assay. TDLN, Cells were isolated from the TDLN of tumor-bearing FVBneuN mice before treatment (day 0), 1 wk after the first treatment (day 7), and 1 wk after the third treatment (day 49). CD8+ T cells were gated on and analyzed for tetramer binding. TIL, Single-cell suspensions were prepared from the tumors of FVBneuN mice on indicated days, CD8+ T cells were gated on and analyzed for tetramer binding. TIL IFN-{gamma} production, Single-cell suspensions obtained from tumors on indicated days were stained for CD8, tetramer binding, and intracellular IFN-{gamma}. Double-positive cells (CD8 and tetramer) were gated on and evaluated for IFN-{gamma} production. All data (except control) are representative of two independent experiments.

 
Beause increased intratumoral T suppressor activity did not appear to affect the ability of CD8+ T cells to produce IFN-{gamma}, we wanted to determine whether this was true for tumor-specific CD8+ T cells as well. CD8+ T cells obtained from tumors on days 0, 7, and 49 were stained for intracellular IFN-{gamma} and analyzed by flow cytometry. The results demonstrate that although the quantity of tetramer-specific T effectors declined over the long term, the remaining cells (on day 49) still responded very strongly to IL-12 plus GM-CSF treatment, with a significantly higher ratio of tetramer-specific cells producing IFN-{gamma} compared with their day 7 counterparts (Fig. 6, TIL IFN-{gamma}-production). This finding essentially mirrored the pattern displayed by the total CD8+ T cell population, further confirming the lack of correlation between the intensity of IFN-{gamma} response and the antitumor efficacy of CD8+ T cells in the repeated vaccination setting.


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
Disclosures
 References
 
These studies demonstrate that the failure of chronic immune therapy to maintain tumor suppression in her-2/neu transgenic mice is not due to an inability to induce immune activation, but rather is associated with the development of an increasingly stronger intratumoral T suppressor cell response, resulting in enhanced blocking of T cell cytotoxicity. Furthermore, augmentation of homeostatic regulatory activity is accompanied with a dramatic loss of tumor-specific CD8+ CTL from the tumor microenvironment in the long term. Thus, these data provide important insights into the question of why repeated vaccination, while able to enhance peripheral antitumor immunity, fails to induce effective tumor eradication.

Quantitative analysis of immune activation and suppression markers in posttherapy tumors demonstrated that intratumoral delivery of IL-12 and GM-CSF promoted a rapid reversal of the suppressive characteristics of the tumor microenvironment. More specifically, treatment enhanced intratumoral IFN-{gamma} production and suppressed Foxp3, TGFbeta, and IL-10 expression. The ability of IL-12 to induce IFN-{gamma}-production by T, NK, and NKT cells is well established (22). Our studies confirmed that augmentation of intratumoral IFN-{gamma}-expression was due, at least in part, to enhanced CD8+ T cell infiltration. The mechanism(s) underlying the reduction in Foxp3, TGFbeta, and IL-10 expression, however, remain to be elucidated. IFN-{gamma} has been shown to inhibit the generation and activation of CD4+CD25+ T suppressor cells (23). Therefore, it is possible that IFN-{gamma} directly mediated the suppression of TGFbeta and IL-10 in the above studies. In contrast, the robust recovery of both TGFbeta and IL-10 on day 7 despite continued high levels of IFN-{gamma} does not support this notion. Alternatively, the treatment-induced decline in Foxp3, IL-10, and TGFbeta expression could be due to a quantitative loss of CD4+CD25+ T cells from the tumor microenvironment and/or a change in their functional characteristics. To this end, we are currently characterizing the posttherapy quantitative and qualitative changes in the tumor-infiltrating CD4+ CD25+ T suppressor population.

Another important finding in this study was that the reversal of intratumoral immune suppression was transient. In fact, T suppressor activity recovered within 1 wk of treatment as indicated by the rebound in intratumoral Foxp3, TGFbeta, and IL-10 mRNA levels. This increase was associated with an upsurge in the numbers of tumor-infiltrating CD4+CD25+Foxp3+ T suppressor cells. The rapid resurgence of suppressor mechanisms between days 4 and 7 indicated that recovery of regulatory activity was tightly linked to effector CD8+ T cell development and that the antitumor CTL had a narrow window of opportunity for eliminating tumors. These findings are consistent with the results of a recent study (24), in which Ag-specific vaccination of tumor-bearing mice resulted in the systemic amplification of adoptively transferred Ag-specific CD4+CD25+Foxp3+ cells. Collectively, these observations support the notion that amplification of T suppressor cells is a critical and highly effective mechanism for homeostatic regulation of posttreatment immune activity within tumors. In contrast, how immune activation leads to a rapid expansion of T suppressors in tumors remains to be determined.

Repeated treatment was able to reverse resurgent suppressor activity; however, this reversion occurred with decreasing efficacy following each treatment. The loss of efficacy correlated with an intensification of the T suppressor rebound. These results correlate well with our previous findings (11), in which repeated treatment resulted in diminished antitumor efficacy in this model. In our previous study, we could not rule out the possibility that the loss of antitumor efficacy was due to a change in the antigenic profile of the tumor. To this end, analysis of her-2/neu expression, which was shown above to be a bona fide target for CTL, did not demonstrate a significant loss of her-2/neu from tumors during repeated treatment (data not shown). These findings are consistent with the hypothesis that the observed loss of therapeutic efficacy during chronic immune stimulation is mediated by a progressive enhancement of the homeostatic regulatory mechanisms rather than the loss of tumor Ag in this model.

Previous studies (22), in numerous laboratories including ours, had established the critical role of IFN-{gamma} in IL-12-mediated tumor eradication providing the rationale for our selection of IFN-{gamma} as a marker for intratumoral immune activation. However, the finding that repeated treatment augmented intratumoral IFN-{gamma}-production but not tumor suppression demonstrated that IFN-{gamma} alone was not sufficient for tumor eradication. This is consistent with the results from a previous report (25), which demonstrated a lack of correlation between intratumoral IFN-{gamma} levels and tumor regression in melanoma patients undergoing peptide vaccination. Furthermore, the above results did not demonstrate a direct antagonistic relationship between intratumoral IFN-{gamma} levels and T suppressor activity, suggesting that intratumoral T suppressor cells were mediating their effect through a mechanism that did not involve suppression of IFN-{gamma}. A recent report (20) showed that T suppressor cells do not necessarily inhibit cytokine production by CD8+ T effectors but mediate their effects via direct suppression of cytotoxic function in a TGFbeta-dependent manner in vivo. Consistent with these findings, analysis of T cell cytotoxic function after one and three treatments demonstrated that multiple treatments resulted in reduced cytotoxic function despite enhanced IFN-{gamma} production. Although T suppressor cells were identified as the most likely instigator of CTL dysfunction in this model, the unusually high levels of intratumoral IFN-{gamma} (>9000-fold above pretherapy) observed after the third treatment raised the possibility that this cytokine itself could be directly involved in T cell inactivation. IFN-{gamma} has been shown (26) to induce the production of IDO by APCs, an enzyme that can mediate T cell anergy via tryptophan catalysis. Therefore, it was also possible that enhanced IFN{gamma} production resulted in IDO-mediated CTL anergy independent of T suppressor activity. Preliminary analysis of posttherapy tumors demonstrated a 5- to 10-fold increase in IDO transcript levels between 6 and 72 h, supporting this notion (data not shown). Recently, T suppressor cells were also shown to induce IDO production by dendritic cells via CTLA-4 ligation (27). Whether enhanced IDO expression was due specifically to IFN-{gamma}, to T suppressor cells, or both, and whether IDO contributed significantly to the loss of CTL cytotoxicity, remains to be determined.

Further analysis of intratumoral CTL activity using tetramer analysis demonstrated a major shift in the clonal profile of the CTL during repeated therapy. Whereas the initial treatment successfully promoted the infiltration of tumors with her-2/neu-specific CD8+ T cells comprising >35% of all CD8+ T cells, the proportion of these cells declined dramatically (<2%) after repeated therapy. Persistent exposure to Ag results in exhaustion/deletion of Ag-specific CD8+ T cells in both chronic virus infection and established tumor models (28, 29, 30). The observation that repeated therapy results in the loss of her-2/neu-specific CD8+ T cells is consistent with Ag overload-mediated exhaustion because her-2/neu expression on tumors was not altered during long-term therapy. Repeated stimulation with cytokine-encapsulated microspheres may in fact accelerate this process. One unanswered question in this study is whether the observed loss of CD8+ T cell cytotoxicity in chronically treated mice is due to enhanced T suppressor activity and/or IDO, to the loss of dominant CTL, or a combination of all of these factors. Studies addressing this question are currently underway in our laboratory. Another interesting question is whether the two mechanisms identified in this study, i.e., augmentation of T suppressor activity and clonal deletion, are independent events or whether intensification of the suppressor response contributes to the loss of her-2/neu-specific CTL from the tumor since T suppressor cells have been shown to possess cytotoxic potential (31). Current evidence from numerous in vitro and in vivo studies (32) does not support a direct role for T suppressors in the control of CTL survival, suggesting that suppression and exhaustion are distinct but complementary events.

The above findings have important clinical implications for the specific approach tested in this study and for cancer immune therapy in general. First, the results demonstrate the importance of the tumor microenvironment in posttherapy immune monitoring as both CD8+ T cells and T suppressors rapidly home to tumors following immune activation. Second, the data establish that posttreatment T cell IFN-{gamma} secretion, a parameter that is commonly used to monitor patient immunity, does not correlate with the efficacy of tumor kill and that monitoring of granzyme/perforin secretion patterns of intratumoral (or TDLN) T cells will likely provide a better correlate. Finally, our studies demonstrate that induction of antitumor immunity is rapidly counteracted by homeostatic regulation, and that repeated stimulation results in a progressive loss of therapeutic efficacy due to increased suppressor activity and eventual immune exhaustion. This finding suggests that standard vaccination protocols have a limited window of efficacy in the established disease setting. Studies (33, 34, 35) have shown that this window can be broadened by blocking homeostatic regulation. On the other hand, although blocking of regulatory mechanisms can dramatically enhance effector activity, this approach does not address the longer-term immune exhaustion issue. Clonal deletion can particularly become a drawback in patients with bulky and persistent disease who require chronic treatment. Therefore, in addition to modulation of immune regulatory mechanisms, approaches targeting multiple Ags in patients with minimal residual disease are likely to provide a more effective vaccine strategy in cancer patients (36).


   Acknowledgments
 
We thank Drs. Elizabeth Jaffee and Todd Armstrong for providing the neu-expressing murine tumor cell lines, anti-neu CTL cell line, and advice on her-2/neu-specific tetramers. We also thank Dr. Stan Wolf of Wyeth for providing the recombinant murine IL-12 and for continued support of our studies.


   Disclosures
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
References
 
The authors have no financial conflict of interest.


   Footnotes
 
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

1 This work was supported by a grant from the Breast Cancer Research Program of the U.S. Army Medical Research and Materiel Command/Department of Defense, Award DAMD17-01-1-0262 (to N.K.E.). Back

2 Address correspondence and reprint requests to Dr. Nejat Egilmez, Delia Baxter Research Building, University of Louisville, Room 119, 580 South Preston Street, Louisville, KY 40202. E-mail address: nejat.egilmez{at}louisville.edu Back

3 Abbreviations used in this paper: Foxp3, Forkhead/winged-helix protein 3; TIL, tumor-infiltrating leukocyte; TDLN, tumor-draining lymph node; IDO, indoleamine 2,3-dioxygenase; FNA, fine needle aspiration. Back

Received for publication February 15, 2006. Accepted for publication April 5, 2006.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 

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