Photodynamic therapy plus low-dose cyclophosphamide generates antitumor immunity in a mouse model

Abstract

Photodynamic therapy (PDT) is a modality for the treatment of cancer involving excitation of nontoxic photosensitizers with harmless visible light-producing cytotoxic reactive oxygen species. PDT causes apoptosis and necrosis of tumor cells, destruction of the tumor blood supply, and activation of the immune system. The objective of this study was to compare in an animal model of metastatic cancer PDT alone and PDT combined with low-dose cyclophosphamide (CY) a treatment that has been proposed to deplete regulatory T cells (T-regs) and increase the immune response to some tumors. We used J774 tumors (a highly metastatic reticulum cell sarcoma line) and PDT with benzoporphyrin derivative monoacid ring A, verteporfin for injection (BPD; 1-mg/kg injected i.v. followed after 15 min by 150 J/cm² of 690-nm light). CY (50 or 150 mg/kg i.p.) was injected 48 h before light delivery. PDT alone led to tumor regressions and a survival advantage but no permanent cures were obtained. BPD–PDT in combination with low-dose CY (but not high-dose CY) led to 70% permanent cures. Low-dose CY alone gave no permanent cures but did provide a survival advantage and was shown to reduce CD4+FoxP3+ T-regs in lymph nodes, whereas high-dose CY reduced other lymphocyte classes as well. Cured animals were rechallenged with J774 cells, and the tumors were rejected in 71% of mice. Cured mice had tumor-specific T cells in spleens as determined by a ⁵¹Cr release assay. We conclude that low-dose CY depletes T-regs and potentiates BPD–PDT, leading to tumor cures and memory immunity.

Despite a long-held realization that cancer leads to an impaired immune response, and speculation that the immune system is capable of mounting an effective antitumor immune response if the correct stimulation is applied, cancer immunotherapy has not yet reached the status of a mainstream therapy (1, 2). Many approaches have been suggested for reversing this ability of tumors to evade the immune system (3), including nonspecific biological response modifiers (4), cytokine therapies (5), cancer vaccine approaches (6), cell-mediated immunotherapies (7), and gene therapy (8). Because it is well known that most deaths from cancer are caused by metastatic tumor, the ideal cancer treatment would be one that not only effectively eradicated the primary tumor, but in addition, simultaneously activated the immune system to attack distant metastases. Although surgery and radiotherapy can often destroy primary tumors with high efficiency, at high doses they are thought to have an immunosuppressive effect, thus allowing micrometastases to grow unchecked.

Photodynamic therapy (PDT) uses a photoactivated dye or photosensitizer (PS) in combination with visible light that produces reactive oxygen species and destroys tumor cells and malignant tissue (9–11). PDT is approved for multiple indications in the United States and many other countries (12). Mechanisms that have been shown to be involved in the tumoricidal effect include direct cytotoxicity to tumor cells (13), shutting down of the tumor vasculature, thus starving the tumor of oxygen or nutrients (14), and the induction of a host immune response (15). The precise mechanisms involved in the PDT-mediated induction of antitumor immunity are not yet completely understood, but advances are constantly being made (16). It is known that after PDT there is an acute inflammatory response in the treated area that rapidly causes a massive regulated invasion of neutrophils (17), mast cells, and monocytes/macrophages (18) that may outnumber resident cancer cells. Most notable is a rapid accumulation of large numbers of neutrophils, which have been shown to have a profound impact on PDT-mediated destruction of tumors, and it has been shown that depletion of neutrophils in tumor-bearing mice decreased the PDT-mediated tumor cure rate (19). The acute inflammatory response is thought to attract dendritic cells that can take up necrotic and apoptotic tumor cells and migrate to lymph nodes (LNs) where they prime tumor-specific cytotoxic T cells (CTLs). The activity of CTLs is not limited to the original PDT-treated site but can include disseminated and metastatic lesions of the same cancer (20).

Reports dating from as early as 1981 (21, 22) have shown that under some conditions low-dose cyclophosphamide (CY) (a traditional cytotoxic cancer drug that damages tumor DNA) can potentiate antitumor immunity in mouse models. Several mechanisms have been proposed to explain this observation, including depletion of suppressor T cells (21), reduction of immunosuppressive cytokines (23), and antiangiogenesis (24). Since the explosion of interest in regulatory T cells (T-regs) expressing CD4, CD25, and the transcription factor Foxp3 (25, 26), it has become accepted that low-dose CY selectively depletes T-regs in mice and thus increases both the priming and effector phases of the antitumor immune response (27).

Here, we report that PDT can be effectively combined with low-dose CY to cure a highly metastatic mouse tumor and produce tumor-specific CTLs and potent memory immunity.

Results

J774 Tumor.

The J774 tumor is a BALB/c mouse reticulum cell sarcoma (macrophage tumor) that is aggressive, invasive, and metastatic as shown by hematoxylin and eosin-stained histology (Fig. 1 A–D). It is always fatal to mice and difficult to cure; its immunogenicity is unknown. After sequential amputations after the first day postinjection, 10 mice were evaluated for survival, and all mice were dead after 48 days (Fig. 1E). The mouse that died first was the one who had the latest surgery (10 days after cell injection). Early surgery therefore predicted longer survival consistent with J774 being a highly metastatic tumor that metastasizes as early as 24 h after injection.

Fig. 1.

H&E histology of tissue sections removed from J774 tumor-bearing mice. (A) Tumor invading thigh muscle after s.c. implantation. (B) Metastatic tumor invading omental fat pad. (C) Metastatic nodule in lung. (D) Metastatic nodule in liver. (Scale bar: 100 μm.) (E) Survival in days of 10 mice after sequential daily amputation of the leg bearing s.c. J774 tumors in individual mice commencing 24 h after tumor injection.

PDT.

All of the PDT-treated mice had a response typical of vascular PDT in their treated tumors with the development of a black eschar at 2–3 days post-PDT and subsequent partial or complete disappearance of the tumor with good local tissue healing.

Mouse Survival.

Nine mice were treated with PDT alone and the survival curves of these mice compared with 10 control J774 tumor-bearing mice is shown in Fig. 2A. The median survival of the PDT group was 49 days, and the median survival of the control untreated mice was 35 days. These two survival curves were statistically different by log-rank test (P < 0.005), but nevertheless all PDT-treated mice died.

Fig. 2.

Kaplan–Meier survival curves of groups of mice. (A) No treatment tumor, n = 10; PDT alone, n = 9; low-dose CY, n = 10; low-dose CY + PDT, n = 15. (B) No treatment tumor, n = 10; PDT alone, n = 9; high-dose CY, n = 10; high-dose CY + PDT, n = 10. Mice were killed when the primary tumor reached 1.5 cm in diameter or their body weight dropped ≥15%.

Fifteen mice treated with the combination of both therapies [benzoporphyrin derivative monoacid ring A, verteporfin for injection (BPD)–PDT plus CY] had a dramatically different outcome from those treated with PDT alone. Fig. 2A shows that the combination group had a long-term (>90 days) survival rate of ≈66%. This survival curve was statistically different from curves in control (P < 0.0001) and BPD–PDT alone (P = 0.0001) groups. This is a very important result when compared with no long-term survivals whatsoever in mice treated with BPD–PDT alone. It was important to study the effect of administering a single low dose of CY alone on mice bearing J774 tumors. Thirteen mice were therefore injected with CY (50 mg/kg i.p.) 7 days after the tumor was implanted. These mice had a median survival of 49 days, and the Kaplan–Meier survival curve is shown in Fig. 2A and was significantly different from the following groups: control (P < 0.0001), and BPD–PDT + low-dose CY (P < 0.0001). There was no difference between the survival of the PDT alone and low-dose CY-alone groups.

To discriminate between the antitumor effect of CY and its immune potentiating activity, we repeated the PDT + CY and CY-alone groups of mice with a high dose (150 mg/kg) rather than a low dose (50 mg/kg) of CY. The Kaplan–Meier survival curves of these mice together with that of PDT alone and no treatment control tumor-bearing mice are shown in Fig. 2B. The median survival of the high-dose CY alone group was 30 days (not significantly different from control tumor-bearing mice), and the median survival of the high-dose CY plus PDT group was 42 days, but these curves were not significantly different from each other or from control tumor-bearing mice. However, PDT combined with high-dose CY gave significantly shorter median survival than PDT alone (P < 0.05).

Tumor Rechallenge.

To test whether the total cures obtained with the combination of low-dose CY plus PDT were caused by activation of the host immune system, we rechallenged the mice with 1 million J774 tumor cells in the contralateral leg. The rechallenge procedure showed the following rates of success: in the case of BPD-PDT + CY five of seven mice rejected the tumor rechallenge (71% success). As a control for tumorigenicity of the J774 cells five of five naive mice injected with the same dose of the same cells grew tumors (zero of five rejections). These two groups were significantly different by Fisher's exact test (P < 0.05). To test the tumor specificity of the rejection of the tumor rechallenge the mice that rejected J774 cells were challenged for a second time with a tumorigenic dose of EMT6 cells. This is a BALB/c mammary sarcoma line that is moderately immunogenic. Seven of seven mice (100%) that had previously rejected J774 cells grew tumors with EMT6 cells demonstrating that the induced immune response is specific to J774 tumors.

CTL Chromium Release Assay.

We carried out a radiochromium release cell-mediated cytotoxicity assay on splenocytes isolated from either cured mice that had rejected a rechallenge with J774 cells, J774 tumor-bearing mice, or naïve mice and compared the cytolysis of specific target cells (J774) or irrelevant nontarget cells (EMT6). Fig. 3 shows that lymphocytes from cured mice that had rejected a rechallenge with J774 cells were able to lyse J774 targets in a effector/target (E/T)-dependent manner with statistical significance at E/T ratios ≥10:1. There was some suggestion of cytolytic activity exhibited by lymphocytes from tumor bearing mice at the highest E/T ratios (100:1). The absence of any cytolysis of J774 cells by lymphocytes from naïve mice or by lymphocytes from cured mice against an irrelevant target (EMT6 cells) demonstrates the tumor specificity of the observed cytolysis.

Fig. 3.

Radiochromium release assay for cell-mediated cytotoxicity carried out by incubating LN cells isolated from either cured mice that had rejected a rechallenge with J774 cells, J774 tumor-bearing mice, or naïve mice. Target cells were either J774 (specific) or EMT-6 (irrelevant) cells that had been loaded with radioactive chromate. The indicated E/T ratios were incubated together for 4 h, and specific ⁵¹Cr release was measured by scintillation counting. Values are means of duplicate determinations from three separate mice, and bars are SD. Significance was determined by ANOVA. P values are ***, < 0.001; **, < 0.01; *, <0.05.

Flow Cytometry for T-Regs.

To elucidate the effects of treatment of the mice with either low-dose or high dose CY, we examined the proportion of T cells from mouse LN that expressed CD4 and intracellular FoxP3 2 days after injection with either PBS, low-dose CY, or high-dose CY. Many reports have shown that the intracellular transcription factor FoxP3 is the most specific measure of T-regs in mice (28). The mean fraction of CD4⁺FoxP3 double positive splenocytes gated on the CD4 population is shown in Fig. 4A. Mice with J774 tumors injected with PBS alone showed a small, but significant, increase in double positive cells compared with naïve mice from 18 ± 1% to 22.7 ± 1% (P < 0.01). J774 tumor-bearing mice injected with 50 mg/kg CY showed a marked decrease in double positive cells down to 5 ± 1% (P < 10⁻⁶), whereas mice bearing J774 tumors injected with 150 mg/kg had a significantly higher percentage of double positive cells (8.1 ± 0.3%) compared with low-dose CY (P < 0.05). As a measure of the total number of lymphocytes we also measured the total number of splenocytes isolated per mouse. The results given in Fig. 4B show that mice with J774 tumors demonstrated a consistent splenomegaly with a ≈50% increase in the total number of splenocytes from 112 million to 165 million (P < 0.005). After low-dose CY the mean number of splenocytes decreased >60% compared with tumor-bearing mice to 66 million (P < 10⁻⁴), and after high-dose CY the number decreased even further to 38 million (P < 0.001 vs. low-dose CY). These results are consistent with low-dose CY killing a major fraction of the overall splenocyte population, but with some selectivity for CD4⁺FoxP3⁺ T-regs, whereas the high-dose CY kills even more splenocytes and lymphocytes, thus making the relative proportion of T-regs appear to increase.

Fig. 4.

Quantification of T-regs and splenocytes. (A) Percentages of CD4⁺FoxP3⁺ double positive LN cells from LNs extracted from mice with or without J774 tumors 3 days after low-dose CY injection. (B) Numbers of splenocytes from whole spleens removed from mice as described above. Values are means of duplicate determinations from three separate mice, and bars are SD. Significance was determined by ANOVA.

Discussion

This study has demonstrated that PDT combined with low-dose CY produces a dramatic improvement in survival and significant numbers of cures in a highly metastatic mouse tumor model. No cures but some survival advantage were seen with either treatment alone, whereas when PDT was combined with high-dose CY there was no additional benefit. There was a long-term memory immunity generated by the combination treatment of BPD–PDT and low-dose CY that allowed cured mice to reject a rechallenge with a tumorigenic dose of J774 cells, and the presence of lymphocytes that specifically caused cytolysis of target cells ex vivo was shown.

The PS that we used, BPD after 15 min, is still localized in the vasculature. It has been shown that “vascular”-type PSs such as BPD have greater effectiveness if illumination is carried out at early time points after injection (29). Under these conditions damage is caused to the tumor by shutdown of vasculature, loss of perfusion, and deprivation of the tumor of oxygen and nutrients normally supplied by the circulation. The cells in the tumor tissues undergo necrosis and apoptosis, and these processes increase the release of tumor antigen that can be recognized and presented to the immune system, leading to the generation of a tumor-specific immune response. Other workers who have studied the induction of antitumor immunity after PDT have used PSs with a longer drug–light interval (20, 30), and these might be less vascular in mode of action. No one to our knowledge has compared vascular and cellular PDT regimens to see which is more efficient in inducing immune response against tumors.

The J774 tumor model has not been studied much in relation to its immunogenicity or to its susceptibility to cancer immunotherapy approaches. Its highly metastatic nature does mean that a fully curative therapy has to involve immune stimulation, as even complete surgical removal as soon as 1 day after implantation did not give a cure. Because these tumor cells are derived from the monocyte/macrophage lineage they do express MHC class II molecules in addition to MHC class I molecules, and therefore they might be able to prime the immune system to recognize their own antigens. Our findings that PDT, in combination with low-dose CY, induces specific CTLs capable of ex vivo cytolysis suggests that effective tumor-rejection antigen(s) do exist in J774 cells but remain to be identified. The existence of possible antigens in J774 cells capable of being recognized by the immune system was reported by Fedoseyeva et al. (31) who showed that J774 tumors growing in BALB/c mice generated specific CD4 cells that recognized peptides derived from mutant p53 protein.

T-regs protect the host from autoimmune disease by suppressing self-reactive T cells and may also block antitumor immune responses mediated by tumor-specific T cells (26). The suppressive effect of naturally occurring T-regs against tumor-specific CD8 T cells has been demonstrated both in vitro (32) and in vivo (33, 34). Local depletion of CD4 T cells inside the tumor led to eradication of well established tumors and development of long-term antitumor memory, suggesting that suppression of antitumor immunity by T-regs occurs predominantly at the tumor site and that local reversal of suppression, even late during tumor development, can be an effective treatment (35).

Whether there is a systemic increase of T-regs in cancer-bearing mice is not yet clearly defined because conflicting data have been reported. In a colon carcinoma model, an expansion of T-regs was observed only in the spleen but not in peripheral blood (36). In our results we found a small, but significant, increase in T-regs in tumor-bearing mice. Depletion of T-regs together with other immunostimulatory approaches, for example, CTLA-4 blockade, has also been tested (37). Because immune responses to malignant tumors often are weak and ineffective, solely depleting T-regs might not always result in tumor regression, therefore combining T-reg depletion with other immunologic interventions, for example, transfer of activated T cells (38) or dendritic cell-based vaccinations (39), have been reported. Several molecules have been implicated in the suppressor mechanism underlying the immunoregulatory function of CD4⁺FoxP3⁺T cells, including cell surface molecules such as CTLA-4 and glucocorticoid-induced tumor necrosis factor receptor (40), and secreted molecules such as TGF-β1 and IL-10 (41).

CY administration especially when carried out at low doses has been suggested to have a specific effect in depleting T-regs (42–44) and has been used as an immunomodulator in several studies with the intention to induce tumor regression and increase the concomitant immunity that can be suppressed by T-regs. In our case both low- and high-dose CY decreased the number of total splenocytes and the proportion of T-regs. However, considering the further decrease in lymphocytes caused by high-dose CY, we suggest that populations of CD4 T-helper (Th) cells and CD8 effector T cells necessary for development of an effective antitumor immunity were depleted by high-dose CY, but not by low-dose CY, and that this difference explains the ineffectiveness of high-dose CY in combination with PDT in inducing immunity. High-dose CY plus PDT actually reduced the survival achieved with PDT alone, suggesting that there is some contribution of the host immune system to the outcome of PDT alone, although not sufficient to lead to cures. It is at present uncertain exactly what is the mechanism for selective toxicity of low-dose CY toward T-regs. Reports have shown a switch from Th2 to Th1 cytokines after low-dose CY (45), inhibition of proliferation in the more rapidly cycling T-regs (46), induction of apoptosis and functional inhibition in T-regs (27), and inhibition of inducible nitric oxide synthase that controls T-reg numbers (47).

PDT has mostly been thought of as a local therapy in which cell and tissue destruction happens primarily in the area that is exposed to light and the immediately surrounding areas. However, mounting evidence suggests that, as opposed to many common cancer therapies that are largely immunosuppressive, PDT can stimulate the immune system (16). In this respect PDT is similar to adjuvant-enhanced laser immunotherapy that has also been shown to be capable of destroying local tumors, while at the same time sensitizing the immune system to destroy distant metastases (48–51). The fact that we observed only long-term cures with the low-dose CY combination treatment suggests that in this tumor model (and perhaps in many others) there is some host factor that is fighting against the immune stimulating effect of PDT. We suggest that this factor could be CD4⁺FoxP3⁺ T-regs because of their susceptibility to low-dose CY and as shown by our flow cytometry results. The effect of low-dose CY alone on the tumor was actually much more pronounced than the effect of high-dose CY alone (compare Fig. 2 A and B). This finding suggests that the effects of CY on J774 tumor are caused by an immunostimulatory effect rather than by the traditional cytotoxic role of CY.

Much work remains to be carried out to conclusively establish the scope of the antimetastatic effect and antitumor immunity effect of the combination of low-dose CY and PDT. Does substitution of low-dose CY with an anti-CD25 antibody produce the same effect in the combination therapy regimen? Does the combination of low-dose CY and PDT work equally well with other less vascular PS (compared with BPD) as the PDT agent? Does the combination work with other poorly immunogenic mouse tumor models? When further work on the role of T-regs in limiting the immune response to human cancer and their role in the immune response after PDT has been carried out in patients, we believe serious consideration should be given to testing T-reg depletion therapies for patients who will receive PDT for cancer especially when it may have already metastasized.

Materials and Methods

Cell Lines and Mouse Tumor Model.

J774 cell line [murine BALB/c macrophage-like reticulum cell sarcoma (52)] was obtained from ATCC and cultured in RPMI media 1640 containing Hepes, glutamine, 10% FCS, 100 units/ml penicillin, and 100 μg/ml streptomycin. Cells were collected for injection by washing with PBS without Ca²⁺ and Mg²⁺ and adding trypsin-EDTA to the plate for 5 min at 37°C. EMT-6 cells, a BALB/c breast sarcoma line (53), were used as an irrelevant target control for radiochromium release assays. All animal experiments were approved by the Subcommittee on Research Animal Care of Massachusetts General Hospital and were in compliance with National Institutes of Health guidelines. Male BALB/c mice (Charles River Labs), weighing 20–25 g, were depilated (Nair; Carter-Wallace Inc.) on the right thigh, and the next day they were injected s.c. with 10⁶ J774 cells suspended in 100 μl of PBS. Tumors grew predictably in all mice and reached a size of 5- to 6-mm diameter in 8–10 days after injection at which time they were used for PDT. In some mice tumors were allowed to progress for 4 weeks, and mice were killed for examination of organs by standard H&E histopathology. Low doses (50 mg/kg) or high doses (150 mg/kg body weight) of CY (Sigma) in sterile PBS was injected i.p. 2 days before the PDT treatment or after 7 days tumor growth in CY-alone groups. In the literature of low-dose CY studies in mice and rats there have been several doses used at both low doses and high doses. For low doses from 10 to 100 mg/kg have been used, and for high doses from 150 to 300 mg/kg have been used (54). Our selection of 50 and 150 mg/kg as low and high dose, respectively, was based on these reports. Mice were anesthetized with an i.p. injection of ketamine/xylazine mixture (90 mg/kg ketamine, 10 mg/kg xylazine) before any invasive procedure (PDT or surgery).

Amputation.

Ten mice with s.c. J774 tumors in the midthigh had their legs sequentially amputated above the injection site on successive days starting at 1 day after injection until day 10. This procedure was done under anesthesia. With a scalpel, the femoral artery was isolated, sutured, and cut with VICRIL 7–0 synthetic absorbable suture (Ethicon Inc.), and the bone was cut after hemostasis. The wound was closed in two planes: first, muscle was closed with VICRIL 7–0 suture, and the second plane was skin closed with black monofilament Nylon nonabsorbable surgical suture (Ethicon, Inc.). All of these procedures were done following aseptic techniques.

PDT.

BPD was a generous gift from QLT Inc. (Vancouver). The lyophilized powder was reconstituted with sterile 5% dextrose injection (Abbott Laboratories). Mice were anesthetized as above, and BPD at 2 mg/kg was injected in 0.1 ml in the lateral tail vein with an insulin syringe (30-gauge needle). Tails were warmed in water (50°C) to facilitate the procedure. PDT was carried out at 15 min after injection of BPD. Illumination was carried out by using a 1-W solid state diode laser emitting light at 690 nm (+/− nm) (B&W Tek Inc.) for activation of BPD. The laser was coupled into a 400-μm fiber via a SMA connector, and light from the distal end of the fiber was focused into a uniform spot with an objective lens (no. 774317; Olympus). The spot had a diameter of 1.2 cm and was positioned so that the entire tumor and a surrounding 2–3 mm of normal tissue were exposed to light. A total fluence of 150 J/cm² was delivered at a fluence rate of 100 mW/cm² as measured with a power meter (model DMM 199 with 201 standard head; Coherent). At the completion of the illumination mice were allowed to recover in an animal warmer until they resumed their normal activity.

Follow-Up of Mice.

Mice were examined three times a week. They were weighed, and the orthogonal tumor dimensions (a and b) were measured with vernier calipers. The tumor volume was calculated according to the formula, volume = 4π/3 [(a + b)/4]³. Tumors were treated when they were 6 mm in diameter (volume ≈110 mm³). Mice were killed when either the primary tumor reached a diameter of 1.5 cm or they had lost 15% or more of their body weight. Mice were considered cured when they were healthy and tumor-free for 90 days and their body weight remained normal. Mice that were cured were rechallenged with tumor (injection of 1 million J774 cells in the contralateral leg) together with naïve mice. Mice that rejected the J774 rechallenge were subsequently challenged with 1 million EMT6 cells.

CTL Chromium Release Assay.

Whole spleens from tumor-immune mice killed 2 days after another J774 rechallenge and from J774 tumor-bearing mice and naïve mice as controls were forced through coarse (70-mesh) steel screens to make single-cell suspensions. Erythrocytes were lysed with red blood cell lysis buffer (Roche Diagnostics). Cells were washed three times with full culture medium, and viable trypan blue-excluding cells were counted on a hemocytometer, allowing the total number of splenocytes isolated per mouse to be calculated. Single-cell suspensions of splenocytes were washed and resuspended in stain buffer (BD Pharmingen). Splenocytes were plated in six-well plates in culture medium for 1 h to allow adherent cells to attach. The supernatant with lymphocytes (nonadherent splenocytes) was collected, and cells were counted and used for further assays. Cytotoxicity was measured by ⁵¹Cr release assay. Nonadherent splenocyte suspension (0.1 ml) as effector cells were dispensed to wells of U-bottom 96-well microtiter plates, with replicates of six wells for each E/T ratio. One million target cells (J774 or EMT6 as irrelevant target) was labeled for 2 h with 100-μCi of ⁵¹Cr (as sodium chromate; PerkinElmer Life and Analytical Sciences) and washed with culture medium and then 10,000 target cells were mixed with effector CTLs at various E/T ratios and incubated for 4 h at 37°C, 5% CO₂. The amount of ⁵¹Cr released into the supernatant by killed target cells was quantitated in a gamma scintillation plate reader (Wallac). The final percentage of specific lysis was calculated as follows: [test ⁵¹Cr released − spontaneous ⁵¹Cr released] / [maximum ⁵¹Cr released − spontaneous ⁵¹Cr released]. The maximal release was obtained by incubation of target cells in 0.5% SDS.

Flow Cytometry for T-Regs.

On the third day after CY administration mice were killed, and inguinal LNs and spleens were removed from naïve mice and tumor-bearing mice with and without CY. LN in 1.5-ml microcentrifuge tubes containing 0.5 ml of ice-cold PBS were homogenized with a pellet pestle motor (Kontes Glass). Spleens were manually homogenized in a ground glass homogenizer in 5 ml of PBS. Homogenates were passed through a 70-μm mesh nylon cell strainer (BD Falcon) to make single-cell suspensions. Splenocytes were counted by trypan blue exclusion on a hemocytometer after red blood cell lysis. T-regs were stained with a mouse regulatory T cell staining kit (no. 88-811540; eBioscience). A total of 10⁶ LN cells were resuspended in 1 ml of fixation/permeabilization solution, incubated on ice for 40 min, washed, treated with Fc receptor blocking antibody, and incubated with FITC-labeled anti-CD4 and phycoerythrin-Cy5-labeled anti-FoxP3 antibodies for 1 h on ice. After washing, LN cells were analyzed by flow cytometry (FACScalibur; BD Biosciences) together with the relevant isotype controls. Thirty thousand total events were counted. FACS scattergrams were analyzed by first gating for size and CD4 expression on FL1 vs. FSC dot plot and next by replotting CD4⁺ cells on FL1 vs. FL3 dot plot to assess the percentage of CD4–FoxP3 double positive cells that was multiplied by FL1 mean.

Statistics.

All values are expressed as ± SD. Survival analysis was performed by using the Kaplan–Meier method. Survival curves were compared, and differences in survival were tested for significance by using a log rank test in the computer program GraphPad Prism. Differences between means of chromium release and FACS analysis were tested for significance by single-factor ANOVA in Microsoft Excel. The tumor rejection frequency was analyzed by Fisher's exact test. P < 0.05 was considered significant.