Cancer Therapy Vol 2, 375-388, 2004

Reversal of immune suppression following vaccination with recombinant vaccinia virus expressing IL-2 in an orthotopic murine model of head and neck squamous cell carcinoma**

Research Article

Santanu Dasgupta¹, Malaya Bhattacharya-Chatterjee¹, Bert W. O’Malley, Jr.², Sunil K. Chatterjee¹*

¹Department of Internal Medicine and the Barrett Cancer Center, University of Cincinnati, Cincinnati, Ohio 45267. ²Department of Otolaryngology Head and Neck Surgery, University of Pennsylvania Health System, Philadelphia, Pennsylvania

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*Correspondence: Sunil K. Chatterjee, Department of Internal Medicine and the Barrett Cancer Center, University of Cincinnati, Cincinnati, Ohio 45267; Telephone: (513) 558-0424; Fax: (513) 558-0505; E-mail: sunil.chatterjee@uc.edu

Key words: head and neck cancer, vaccinia virus, interleukin-2, immune suppression

Abbreviations: Dendritic cells, (DC); Head and neck squamous cell carcinoma, (HNSCC); Immunomodulatory molecules, (IMM); Inducible nitric oxide synthase (iNOS); Phosphate buffered saline (PBS); plaque forming units (pfu); Proliferating Cell nuclear antigen (PCNA); Recombinant vaccinia virus, (rvv); Tumor draining lymph nodes, (TDLN); Vascular Endothelial Growth Factor Receptor (VEGF-R)

**This work was supported in part by the grant RO1-CA 89748 from the National Cancer Institute.

Received: 20 September 2004; Revised: 20 October 2004

Accepted: 22 October 2004; electronically published: October 2004

Summary

Immune cells from mice bearing SCC VII/SF tumors cells in the oral cavity did not respond to mitogenic stimulation. The immunosuppression was mediated by a number of immunomodulatory molecules (IMM), such as GM-CSF, IL-10, TGF-b1, iNOS and VEGF. IMM had been shown to be immunosuppressive in a significant number of cancer patients, including patients with HNSCC. A single subcutaneous injection of irradiated SCC VII/SF tumor cells, infected with a recombinant vaccinia virus expressing IL-2 (rvv-IL-2) reversed the expression of the IMM, both at the mRNA and protein levels, at least partially and restored the anti-tumor cellular immunity. Assay of cytokines from the immune cells on stimulation by SCC VII/SF tumor cells suggested the induction of Th1 type helper activities by the subcutaneous vaccination. Mature dendritic cells (DC) were recruited in the tumor sites and large numbers of both CD4+ and CD8+ T cells were detected near DC following the subcutaneous vaccination. The numbers of PCNA positive cells were reduced as a result of vaccination, suggesting inhibition of tumor cell proliferation. IMM inhibited the expression of MHC class I molecules, which were restored by the subcutaneous vaccination. The numbers of VEGF receptors (VEGF-R) were also reduced by subcutaneous vaccination. Subcutaneous immunization with irradiated SCC VII/SF cells, mixed with recombinant IL-2 induced Th1 helper and cytotoxic T cell activities, although these activities were lower compared to vaccination with rvv-IL-2 infected irradiated SCC VII/SF tumor cells. We concluded that a single subcutaneous injection with irradiated tumor cells infected with rvv-IL-2 or mixed with rIL-2 can reverse the immunosuppression mediated by IMM and restore tumor specific immunity.

Squamous cell carcinoma of the head and neck (HNSCC) is the most common malignancy of the upper aero digestive tract, accounting for more than 90% malignancies in this area (Chen et al, 2003). The annual incidence of the disease is alarmingly high with 500,000 new cases worldwide (Kim et al, 2002). It is the fifth leading cause of cancer incidence and sixth leading cause of cancer related death (Kim et al, 2002). Despite significant improvement in surgery, chemotherapy and radiation therapy, the overall survival for HNSCC patients has not improved in the last 30 years (Kim et al, 2002; Bray et al, 2003). It appears that with the currently applied methods for loco-regional treatment, small undetectable tumor deposits remain in the primary tumor site, resulting in local and distant relapse within a short period of time after primary therapy (van Dongen et al, 1996). As a result the overall 5-year survival rate remained at 50%, with only a 30% 2-year survival rates for cancer stages III and IV (Bray et al, 2003). Thus, there is an urgent need for an effective adjuvant therapy for the eradication of residual disease after initial conventional therapy for HNSCC patients. Immuno/gene therapy represents a promising treatment strategy for these patients with minimal residual disease. HNSCC are ideal for immunological study as they are visible, easily biopsed and often removed with their regional lymph nodes (Hadden, 1997).

Immunotherapy for cancer has been shown to be effective for only a small population of patients with melanoma and renal-cell carcinoma (Rosenberg et al, 1989). Melanoma and renal-cell carcinoma seem to be among the most immunogenic of human cancers, as evidenced by their high rate of spontaneous regression (Mastrangelo et al, 1975). Immunotherapy of HNSCC is a greater challenge, as patients with HNSCC are highly immunosuppressed (Vokes et al, 1993; Hadden et al, 1994; Gooding et al, 1995) in spite of the fact that these tumors are generally infiltrated with T lymphocytes (Heo et al, 1987; Whiteside et al, 1987). T cells from these patients show only partial response to mitogenic stimulation (Wanebo et al, 1975; Avradopoulos et al, 1997), suggesting that these cells are functionally defective. It has been proposed that the administration of T cell growth factor, such as IL-2 or INF-g may restore the T cell defect (Hamasaki and Vokes, 1995). Although systemic administration of rIL-2 in HNSCC patients caused little or no response, administration of natural or rIL-2 at the tumor site or around TDLN showed initial response. Unfortunately, all patients had recurrent tumor after an interval of 3-5 months and were refractory to further IL-2 treatment (De Stefani et al, 2002). Local administration of IL-2 probably was less effective for therapy of HNSCC patients due to the short half-life of this cytokine (Konrad et al, 1990). Continuous generation of IL-2 at the tumor site may be more effective.

One approach to continuous delivery of IL-2 would be the use of recombinant replication-competent viruses expressing the desired cytokine. In addition, lytic viruses, such as vaccinia, may release potential tumor-associated antigens at the tumor sites and make the antigen available to the antigen presenting cells. Vaccinia virus is a strong immune adjuvant and has been shown to invoke humoral and cellular immunity against the transgene in numerous studies in animal and patients (Moss and Flexner, 1987). An additional advantage of using a recombinant vaccinia virus (rvv) for this purpose is that replication-competent virus can be injected directly into tumors. Moreover, a number of studies demonstrated that vaccinia virus is capable of infecting and replicating in tumor lesions despite the presence of systemic neutralizing antibodies (Tsang et al, 1995; Robinson et al, 1998; Mastrangelo et al, 1999; Chen et al, 2001). We demonstrated the anti-tumor efficacy of rvv expressing the cytokines GM-CSF and IL-2 in a number of animal models (Qin and Chatterjee, 1996a,b,c; Chatterjee et al, 1999; Qin et al, 2001; Dasgupta et al, 2003). The safety of vaccinia virus in healthy population has been well established by the smallpox eradication program. Rvv has been also used safely in cancer patients (Tsang et al, 1995; Mastrangelo et al, 1999). In spite of that since HNSCC patients are severely immunocompromised, we are exploring the potential of this vaccine in an orthotopic model of head and neck cancer (SCC VII/SF) before translation to HNSCC patients.

In order to translate results obtained in animal models to patients it is necessary that the model closely resemble the human disease. SCC VII/SF tumor model has the confirmed tumor histological characteristics of squamous cell carcinoma, the most common tumor in the head and neck region. The actual anatomical site and the initial loco-regional aggressiveness of this tumor, with early direct extension into the neck and later cervical lymph node and pulmonary metastasis, resemble the biological behavior of the tumor progression seen in HNSCC (O'Malley et al, 1997). Moreover, like human HNSCC immune cells isolated from the tumor bearing mice do not respond to mitogenic stimulation (Qin et al, 2001). Thus, the results with this model are likely to be translated for development of therapy for HNSCC.

In earlier studies using this model we demonstrated that a combined subcutaneous vaccination with irradiated tumor cells, infected with rvv expressing IL-2 (rvv-IL-2) plus intratumoral rvv-IL-2 injection resulted in enhanced survival and tumor regression compared to intratumoral rvv-IL-2 injection alone (Dasgupta et al, 2003). Irradiated whole tumor cells for subcutaneous vaccination were used to provide tumor-associated antigens at the injection site. While intratumoral injection was performed for the expansion of T cells in the tumor bed by IL-2 from rvv-IL-2 (Dasgupta et al, 2003). The subcutaneous immunization induced helper as well as cytotoxic T cell activities. However, complete eradication of tumors was not achieved by this treatment (Dasgupta et al, 2003) probably due to the presence of immune suppression. In this study we investigated the mechanism of reversal of immune suppression and resulting enhanced anti-tumor immunity induced by the subcutaneous immunization so that vaccination strategy can be improved and used for complete eradication of tumor.

II. Materials and methods

A. Animals and reagents

We obtained 6-8 week old female syngeneic C3H/HeJ mice form the Jackson Laboratory (Bar Harbor, ME, USA). We purchased tissue culture media and reagents from Life Technologies, Inc. (Gaithersburg, MD, USA). We bought recombinant human IL-2, purified rabbit anti-mouse anti-iNOS and anti-rabbit IgG antibodies from Chemicon International Inc. (Temecula, CA, USA) and murine GM-CSF from BD Pharmingen (San Diego, CA, USA). We also obtained the following anti-mouse monoclonal antibodies from BD Pharmingen: purified anti-CD4 (clone RM4-5), anti-CD8 (clone 53-6.7), CD11c (clone HL3), PCNA, Flk-1 (clone Avas 12a1), biotin-anti-H-2K^k (clone 36-7-5), biotin-anti-IgG. We bought cytokine ELISA kits for mouse TGF-b1, GM-CSF, IL-10, IL-2, IFN-g, IL-4 and IL-5 from R&D Systems Inc. (Minneapolis, MN, USA). For the lysis of erythrocytes, RBC lysis buffer (Tris ammonium Chloride, pH 7.5) was purchased from Sigma Chemical Co. (St. Louis, MO, USA). All PCR primers were procured from Integrated DNA Technologies (Coralville, IA, USA).

B. Recombinant vaccinia virus

We used two rvv, rvv-IL-2 expressing human IL-2 and rvv-lacZ expressing Escherichia coli b-galactosidase for the preparation of the vaccines. The procedures for the preparation of rvv-IL-2 and rvv-lacZ have been described previously (Qin and Chatterjee, 1996c). For mock vaccination, we used 100ml of PBS and injected it into the flank or at the tumor site of the mice.

C. Cell line and tumor development

We cultured murine SCCVII/SF, AG104A and C15 tumor cells in Dulbecco’s modified Eagle’s medium containing 10% fetal calf serum, 100 units/ml penicillin, 100mg/ml amphotericin B, and 2 mM L-glutamate. For the development of oral tumor, we anesthetized syngeneic C3H/HeJ mice with 3% Isoflurane (Abbott Laboratories, Chicago, IL) and then injected live SCCVII/SF cells (1 x 10⁵) slowly into the floor of mouth with a 23-gauge needle at the depth of mylohyoid muscle (Qin et al, 2001; Dasgupta et al, 2003). This resulted in tumor development (~40-50 mg) in the floor of the mouth in 5-7 days (Dasgupta et al, 2003). All procedures were performed in accordance with the University of Cincinnati Institutional guidelines for the care and use of laboratory animals.

D. Treatment of mice by subcutaneous immunization with tumor cells

We irradiated monolayer of SCC VII/SF cells with a total of 10,000 rad of g-radiation (Cesium-167). Irradiated cells (1X10⁴) were mixed with 125 units of rIL-2 or GM-CSF in a total volume of 100ml of phosphate buffered saline (PBS) for subcutaneous injection into the left flank of the mice. When rvv were used for vaccine preparation, the cells were infected with 1X10⁵ plaque forming units (pfu)/ml of rvv. After 24 h of infection, we injected 1X10⁴ cells in 100ml of PBS as described above. Control vaccines were prepared using rvv-lacZ. For mock vaccination we used 100ml of PBS. We prepared and transplanted the tumor cells as above on day 0. Vaccination was given on day 7 when tumor weight reached ~ 40- 50 mg. Tumors and immune cells from spleen and TDLN was isolated on day 9.

E. RT-PCR analysis of immunomodulatory molecules

Total RNA prepared from tumor tissues of vaccinated mice was subjected to RT-PCR as described earlier (Qin et al, 2001). The reaction was carried out in a 50ml volume using Qiagen one step RT-PCR kit (Qiagen, Valencia, CA). The sequence of the primers are: GM-CSF: forward 5’-CCCGCTCACCCATCACTG-3¢, reverse 5¢-GGACTGGTTTTTTGCATTCAAAGG-3¢, IL-10: forward 5¢-GTACAGCCGGGAAGACAA-3¢ reverse 5¢-TTTGATCATCATGTATGCTTC-3¢, TGF-b1: forward 5¢-GAGGTACCGCCCGGCCCG-3¢, reverse 5¢-GGTTCAGCCACTGCCGTA-3¢, iNOS: forward 5¢-GGCCCCTGGTAGACCTCAGCT-3¢, reverse 5’-CCCACCGGTGAGGATGCTCAA-3¢ and VEGF: forward 5¢-GCCCTGGAGTGCGTGCCCACGTCAGAGAGCA-3¢, reverse 5¢-TGGCGATTTAGCAGCAGATA-3¢. b–Actin was used as internal control. Amplified DNA fragments were analyzed by electrophoresis in 1.5% agarose gels.

F. Real-time RT-PCR analysis of the immunomodulatory molecules

For quantitative analysis, total RNA extracted was subjected to real-time RT-PCR using Cepheid Smart Cycler system (Sunnyvale, CA) in triplicate. Same primer pairs as above were used for these experiments. Before real-time analysis of the target genes, expression level of the internal control b-actin in all samples was normalized with respect to the threshold cycle number (Ct). Relative fold number of the target gene was then expressed with respect to the normalized internal control b-actin in each case. PCR reaction was carried out in 20ml reaction volume using SYBR Green QuantiTect RT-PCR kit (Qiagen, Valencia, CA, USA). We set the reaction condition according to the manufacturer’s instructions. Briefly, reverse transcription was carried out at 50⁰C for 20-30 min. Inactivation of Reverse Transcriptase, HotStarTaq DNA polymerase activation and template cDNA denaturation were carried out at 95°C for 15 min. The final cDNA amplification step comprised of 40 cycles at 94°C for 30 s, 52°C for 30 s and 72°C for 1 min. Data analysis was done using 2^-dd Ct method (Pfaffl, 2001; Miyazawa et al, 2004). Relative fold number of the target gene expression was determined by subtracting the Ct value of the reference gene from the Ct value of target gene in all cases.

G. Preparation of tumor homogenate

We excised the tumors and removed the necrotic parts. Tumor (20 mg) from each vaccinated mice were excised into small pieces and washed thoroughly to remove as much as possible infiltrating immune cells. Single cell suspension of tumors were made by mechanical dispersion and incubated for 4-5 h at 37°C to remove the remaining non-adherent immune cells. Adherent tumor cells were gently scrapped and homogenized by using a hand-held homogenizer. Following a brief centrifugation at 500g for 5 min, we harvested the clear supernatant for assay of the IMM by ELISA.

H. ELISA of immunomodulatory molecules from tumor homogenate and cytokines from immune cells following vaccination

Levels of TGF-b1, GM-CSF and IL-10 in the tumor tissue homogenate of the vaccinated mice were determined using ELISA kits. We isolated splenocytes and immune cells from TDLN from mice as described earlier (Dasgupta et al, 2003) on day 9 after one time vaccination as described above. Briefly, we crushed the tissues and then passed the materials through 70 mm nylon mesh and collected the cells in culture medium (RPMI-1640). Following two washes in the culture medium, we incubated the cells for 10 min in RBC lysis buffer. Levels of cytokines secreted by the immune cells following stimulation with SCC VII/SF cells were determined using ELISA kits. All experiments were performed in triplicate. The lower limit for the cytokine detection was 5pg/ml.

I. Immunohistrochemistry

Fresh frozen tumor sections (5m) from vaccinated mice were fixed in 1% para-formaldehyde for 10 min. After washing 3 times in PBS, slides were incubated in 0.3% H₂O₂ followed by blocking with rabbit or mouse serum for 15 min. For staining of VEGF-R and iNOS, we incubated for 1 h tumor sections with purified anti-mouse Flk-1 and anti-iNOS antibodies respectively, followed by extensive washing with PBS. Biotin labeled goat anti-rat IgG and anti-rabbit IgG were used as secondary antibodies for Flk-1 and iNOS respectively. After extensive washing with PBS and 30 min incubation in pre-diluted streptavindin-HRP, we stained the sections with freshly prepared DAB solution for 5-10 min and rinsed with water. Sections were counterstained in Harry’s hematoxyline. Matched isotypic controls were used in each case. We repeated each experiment at least 3 times. For staining of anti-C3H/HeJ mouse MHC class I molecules, we incubated sections in biotin labeled anti-mouse H-2K^k antibody and stained and counterstained as described above. For counting of the positively stained cells or intensity measurement we used Metamorph software (Universal Imaging, Downington, PA). At least 10 fields were chosen at random for counting and data are expressed as mean±SE. For intensity measurement lowest value (£50.00) was represented by a single + sign and each fold increase was represented by additional + sign.

J. T cell proliferation assay

Immune cells were isolated as described above. We placed 2 x 10⁵ cells in each well of a 96-well flat bottom tissue culture plate in a volume of 100 ml. We added irradiated SCC VII/SF or control tumor cells (1x10⁴/50 ml) as stimulators to each well. Irradiated splenic macrophages at a concentration of 3x10⁴/50 ml was used as antigen presenting cells. We mixed the contents gently and incubated the plates for 5 days in a tissue culture incubator. We pulsed the cells with 1 mCi of [³H] thymidine 18 h before harvesting. We performed each assay in triplicate and calculated the mean from samples with SD of less than 10%. We determined the stimulation indices by dividing the mean counts per min of each sample by the counts in the medium without the stimulant. For positive control we used Concavalin A at a concentration of 200 ng/well. Other controls were irradiated fibrosarcoma cell line AG104A (H-2K^k) and colorectal carcinoma cell C15 (K-2^b).

K. Assay of cytotoxic T cell activity

We prepared the splenocytes and immune cells from TDLN as described above. Following washes in CTL medium (AMV VI, Invitrogen) we placed 4x10⁶ cells/well contained in 1 ml culture medium in the wells of a 24-well culture plate. We added as stimulator 1x10⁵ irradiated SCC VII/SF or other relevant target cells in 1 ml medium per well. We added human rIL-2 (1ng/well), mixed the contents gently and incubated the plates for 7 days in a tissue culture incubator. For the preparation of the target cells we labeled 1x10⁶ cells with 200 mCi of [⁵¹Cr] and incubated at 37°C for 1 h. We washed the cells 4-5 times with cold medium and adjusted the volume to have a target cell concentration of 4x10⁴/ml. We placed 100 ml of labeled target cells in a well of 96-well round bottom plate. We added immune cells (effector) to obtain an effector/target ratio 50:1 and 100:1 in a final volume of 200 ml. Target cells were SCC VII/SF (H-2K^k), AG104A fibrosarcoma (H-2K^k) and colorectal carcinoma cells, C15 (K-2^b). For the calculation of spontaneous release, we added 100 ml of medium instead of effector cells per well and for the calculation of maximum release we added 5% Triton-X-100. Spontaneous release from all cell lines was less than 15%. For the lysis of the tumor cells, we incubated the plates for 6 h in a tissue culture incubator. We counted the radioactivity released in the aliquots of the incubation mixture by using a g counter. To calculate the percentage of specific lysis we used the formula [(sample counts-spontaneous release/maximum release-spontaneous release)]x 100. We performed each assay in triplicate and repeated the experiment at least two times.

L. Statistical analysis

We used Student’s t test for normally distributed variables. When the data did not fulfill the criteria of being normally distributed, we used non-parametric statistics (Mann-Whitney rank sum test). We performed all statistical evaluation using SigmaStat software (Jandel, San Rafael, CA) and considered P< .05 to indicate statistical significance.

III. Results

A. Expression in tumor microenvironment of mRNA encoding the immunomodulatory molecules and reversal by subcutaneous vaccination with irradiated, rvv-IL-2 infected tumor cells

We demonstrated previously that immune cells from mice bearing SCC VII/SF tumors did not respond to mitogenic stimulation (Qin et al, 2001). Tumor induced immune suppression often is caused by the secretion of various immunomodulatory molecules (IMM) in tumor microenvironment (Kehrl et al, 1986; Mills, 1991; Pisa et al, 1992; Young et al, 1996a, b; Hsieh et al, 2000). In order to investigate whether tumor microenvironment in this model contains similar IMM, we compared the levels of expression of a number of such molecules in cultured SCC VII/SF cells and tumor lesions by RT-PCR. Levels of IL-10, GM-CSF, TGF-b1 and iNOS were significantly higher in tumor lesions compared to the cultured cells (Qin et al, 2001). Since a single subcutaneous injection of irradiated, rvv-IL-2 infected SCC VII/SF cells restored T helper as well as cytotoxic activities (Dasgupta et al, 2003), we examined whether this immunization also inhibited the expression of the IMM.

Levels of the expression of mRNAs encoding GM-CSF, IL-10, TGF-b1, VEGF and iNOS in tumor homogenates from vaccinated mice were compared by semi-quantitative RT-PCR. Mice were vaccinated once with PBS or irradiated, rvv (rvv-lacZ or rvv-IL-2) infected SCC VII/SF tumor cells. RT-PCR products were separated by agarose gel electrophoresis and the results are shown in Figure 1A. The levels of mRNAs encoding GM-CSF, iNOS and VEGF in tumors of mice treated with rvv-IL-2 (lane 3) were lower compared the rvv-lacZ (lane 2) or PBS treated mice (lane 1). Band for IL-10 was undetectable in the tumors of mice treated with rvv-IL-2 (lane 3).

For quantitative determination of the levels of mRNAs encoding IMM, we performed Real-Time RT-PCR and the results are shown in Figure 1B. Level of expression of GM-CSF mRNA was 1/4 in tumor from mice treated with rvv-IL-2 compared to rvv-lacZ and 1/6 compared to the PBS treated mice. Expression of IL-10 was negligible, almost undetectable in rvv-IL-2 treated mice. Levels of VEGF and iNOS were also reduced by vaccination with rvv-IL-2 (1.6-fold), although not as much as GM-CSF. These results suggested that subcutaneous immunization with irradiated, rvv-IL-2 infected SCC VII/SF cells caused significant reversal of expression of GM-CSF and IL-10 and partial (~40%) reversal of VEGF and iNOS in the tumor microenvironment. The expression of mRNA encoding TGF-b1, however, remained almost unaltered (<20% inhibition).

B. Reduced synthesis of immunomodulatory proteins in tumor homogenate from mice following subcutaneous vaccination with irradiated, rvv-IL-2 infected tumor cells

Due to differential degradation of mRNA in tissues, the levels of mRNA encoding a protein may not reflect the levels of the encoded protein. We therefore determined the levels of immunomodulatory proteins in the tumor microenvironment. Levels of TGF-b1, GM-CSF, IL-10 and VEGF were determined in tumor homogenate by ELISA and the results are summarized in Table 1. Although the levels of mRNA encoding TGF-b1 were similar in 3 groups of treated mice (Figure 1) TGF-b1 protein was reduced by more than 3-fold in the tumor homogenate from mice treated with subcutaneous injection with irradiated, rvv-IL-2 infected SCC VII/SF tumor cells compared to PBS treated mice. Levels of GM-CSF were also reduced by 4-fold when the vaccine was prepared by rvv-IL-2. Levels of IL-10 in this group of mice were negligible at the detection level of 8 pg/ml. Levels of VEGF was reduced by a factor of about 8 by treatment with the rvv-IL-2 vaccine. Except IL-10 and VEGF, which was reduced by about 30% and 50% respectively, the levels of other IMM proteins remained unchanged in mice treated with the control vaccine prepared with rvv-lacZ.

Since no antibody suitable for ELISA of iNOS was available, the expression of iNOS was evaluated by immunohistochemistry of sections of tumors following vaccination. Results shown in Figure 2 demonstrate significantly reduced levels of iNOS in

Figure 1. Expression of immunomodulatory molecules by the oral tumors of vaccinated mice. Total RNA was isolated from oral tumors on day 9 and subjected to conventional (A) and Real-Time RT-PCR analysis (B). b-Actin was used as internal control in the PCR reaction. In the conventional PCR assay (A), expression of GM-CSF, iNOS and VEGF was low in the rvv-IL-2 vaccinated mice (lane 3) compared to the rvv-lacZ (lane 2) or PBS (lane 1) group. Expression of IL-10 was undetectable in the rvv-IL-2 vaccinated mice after 40 cycles of PCR reaction. No marked difference in the expression of TGF-b1 was noted between the groups. By Real-Time RT-PCR analysis, relative fold expression of these genes with respect to control b-actin was determined and values were expressed as mean±SE of triplicate reaction. Expression fold level of GM-CSF, iNOS and VEGF was significantly lower and the level of expression of IL-10 was undetectable in the rvv-IL-2 vaccinated mice compared to the control rvv-lacZ or PBS group (P< .005). No significant fold difference in the expression of TGF-b1 was noted among the vaccinated groups.

Table 1. Assay of immunomodulatory proteins in the tumor homogenate following vaccination

IMM proteins^a

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Vaccine groups GM-CSF IL-10 TGF-b VEGF

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PBS 124±13 87±7 2915±133 571±27

Rvv-lacZ 137±19 59±4 3097±142 256±22

Rvv-IL-2 29±8 8±2 937±89 75±7

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^aAmounts of immunomodulatory molecules (pg/ml±SE) in the tumor tissue homogenate were determined by ELISA. Homogenates were prepared from fresh tumor explants (20 mg) and equal amounts of cell free supernatant were used for ELISA. Details are described in Materials and Methods.

Figure 2. Expression of iNOS in the vaccinated mice. Five-mm fresh frozen serial tumor sections on day 9 were stained with anti-mouse iNOS antibody and staining intensity of the positive cells was measured using Metamorph software from 10 randomly selected fields. Data were converted to mean±SE value. Lowest intensity value corresponds to single + sign and each fold increase was denoted by additional + sign. Expression of iNOS was significantly lower (+) in the rvv-IL-2 vaccinated mice compared to the control rvv-lacZ or PBS treated group (+++).

tumor microenvironment following one vaccination with irradiated tumor cells, infected with rvv-IL-2. Vaccination using the control rvv-lacZ resulted in minor reduction of protein expression in the tumor microenvironment. From these results we conclude that by a single subcutaneous vaccination with irradiated, rvv-IL-2 infected SCC VII/SF cells, suppression of tumor-induced immunity can be reversed.

C. Secretion of cytokines in vitro by immune cells from vaccinated mice on stimulation with irradiated SCC VII/SF tumor cells

We reported previously that subcutaneous injection of irradiated, rvv-IL-2 infected cells induced tumor specific helper and cytotoxic T cells (Dasgupta et al, 2003). For anti-tumor immunity, induction of Th1 subtype of helper cells are necessary. To investigate whether Th1 cells were induced by these vaccines, we determined the types and levels of signature cytokines secreted in the culture media by the immune cells following stimulation with irradiated SCC VII/SF tumor cells. Immune cells were isolated from spleen and TDLN cells of the vaccinated mice and the amounts were determined by ELISA. The results are summarized in Table 2. The amounts of Th1 cytokines, IL-2, INF-g secreted by splenocytes or immune cells from TDLN were significantly higher when isolated from mice treated with subcutaneous injection of irradiated, rvv-IL-2 infected SCC VII/SF cells compared to those secreted by immune cells from PBS treated mice (P <. 001). Th2 cytokines, IL-4, IL-5 and IL-10, on the other hand, were significantly lower (P<.03) in these mice compared to PBS treated mice. Th1 cytokines secreted by immune cells from mice treated with control rvv-lacZ vaccine were also higher compared to those from PBS group however the differences between these two groups were lower. Except IL-10 from splenocytes and TDLN cells and IL-4 from splenocytes, levels of Th2 cytokines secreted by the immune cells from mice treated with rvv-lacZ vaccine were higher. From these results we concluded that a single subcutaneous immunization of mice with irradiated, rvv-IL-2 infected SCC VII/SF cells resulted in induction of mostly tumor specific Th1 type of helper cells. Induction of some Th1 type cells by vaccination with rvv-lacZ probably also took place.

D. Mechanism of action of IMM in immune suppression

Chronic GM-CSF production by tumors suppressed antigen specific CD8+ T cell responses in mice as well as cancer patients. GM-CSF induced suppression of immunity is most common in HNSCC (Young et al, 1996a, b; Bronte et al, 1999). The immunosuppression by GM-CSF is due to the accumulation of immature myeloid cells (Young et al, 1996a, b; Kusmartsev and Gabrilovich, 2002). IL-10 (De Smedt et al, 1997; Girolomoni and Ricciardi-Castagnoli, 1997), TGF-b1 (Albini et al, 1987; Young et al, 1996b; Doran et al, 1997), iNOS (Young et al, 1996b) and VEGF (Oyama et al, 1998; Saito et al, 1998) all suppress the maturation of dendritic cells and suppress anti-tumor immunity. Immune suppression by secreted IMM could be mediated by the effect of these factors on DC. To investigate this possibility we immunostained serial sections of tumors by antibodies specific for mature DC (anti-CD11c) and anti CD4 and CD8 antibodies. Results presented in Figure 3 demonstrate significantly larger number of DC in to the tumor beds of mice vaccinated with rvv-IL-2 (88±6, range 59-114, P< .0001) compared to rvv-lacZ (11±2, range 3-17) or PBS vaccinated group (7±2, range 0-14). Number of CD4+ T cells adjacent to DC in tumor sections from mice treated with the rvv-IL-2 vaccine was also significantly higher (142±12, range 98-211, P< .0001) compared to rvv-lacZ (24±4, range 9-41) or PBS vaccinated group (10±2, range 9-17). This situation is likely to facilitate proper antigen presentation and anti-tumor activities. Similar to the CD4+ T cells, infiltrating CD8+ T cells were also significantly higher (79±8, range 34-129, P < .0001) into the tumor beds of rvv-IL-2 vaccinated group compared to the control rvv-lacZ (17±2, range 4-34) or PBS group (11±2, range 2-27). To determine anti-tumor activities of the T cells, we stained serial sections of tumors with anti-bodies specific for proliferating cell nuclear antigen (PCNA). PCNA is a marker reflecting the activity of cell proliferation, which is closely related to invasion and metastasis of malignant cells (Bantis et al, 2004; Kimos et al, 2004). Numbers of PCNA positive tumor cells in the tumor sections were drastically reduced by immunization of mice with the rvv-IL-2 vaccine (10±2, range 0-18, P< .0001) compared to rvv-lacZ (39±5, range 19-61) or PBS (46±4, range 30-61) group. These data suggested that rvv-IL-2 vaccination resulted in the recruitment and maturation of DC leading to inhibition of tumor cell proliferation.

IMM such as IL-10 may also protect tumors from cytotoxic T cells by down regulation of MHC class I molecules (Salazar-Onfray et al, 1997; Yue et al, 1997; Zeidler et al, 1997). Cultured SCC VII/SF cells, however, express MHC class I molecules (Qin et al, 2001). To investigate whether loss of MHC class I molecule is a consequence of IL-10 secretion in tumor lesions in this model we evaluated the expression MHC class I molecule in fresh frozen tumor sections by immunohistochemistry. Results presented in Figure 4 demonstrate significantly lower number of MHC class I positive cells in the PBS (36±6, range 13-80) or rvv-lacZ (45±8, range 12-89) treated mice compared to the rvv-IL-2 (179±11, range 103-233 P < .0001) vaccinated mice. Notably, numerous

Figure 3. Distribution of DC, CD4+/CD8+ T cells and PCNA into the tumor beds of vaccinated mice. Five-mm fresh frozen serial tumor sections on day 9 were stained with anti-mouse CD11c, CD4, CD8 and PCNA antibodies. Counting of positive cells was carried out using Metamorph software from 10 randomly selected field and data were converted to mean±SE. Number of CD11c, CD4 and CD8 positive cells was always significantly higher into the tumor beds of rvv-IL-2 vaccinated mice compared to the control rvv-lacZ or PBS vaccinated mice (P < .0001). PCNA positive cells were significantly low in number in the rvv-IL-2 vaccinated mice compared to the controls (P < .0001). ® indicated positively stained cells.

Table 2. Secretion of cytokines by immune cells following subcutaneous injection of rvv infected tumor cells.

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Cytokines^a

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Source of IL-2 IFN-g IL-4 IL-5 IL-10

Immune Groups

Cells

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Spleen PBS 49±7 125±46 100±21 48±15 122±11

Spleen rvv-lacZ 60±3 576±39 52±19 68±9 58±3

Spleen rvv-IL-2 177±7 1567±57 34±3 19±2 29±7

TDLN PBS 36±3 13±1 35±7 45±6 412±20

TDLN rvv-lacZ 2±9 44±13 60±9 131±10 50±11

TDLN rvv-IL-2 120±10 30±1 17±2 45±3 23±9

__________________________________________________________________________________________________

^aAmounts of cytokines (pg/ml±SE) secreted by the immune cells after stimulation with irradiated SCCVII/SF cells were determined using ELISA kit. Immune cells were isolated from spleen and TDLN as described in Materials and Methods. Mice were vaccinated on day 7 with PBS and irradiated SCCVII cells infected with rvv-lacZ or rvv-IL-2.

Figure 4. Expressions of MHC class I (H-2K^k) and VEGF-R in the vaccinated mice. Five-mm fresh frozen serial tumor sections on day 9 were stained with anti-mouse MHC class I (H-2K^k) and anti-VEGF-R (flk-1) antibodies. Counting of positive cells or measurement of staining intensity was carried out using Metamorph software from 10 randomly selected fields. In case of positively stained cells, data were converted to mean±SE and for intensity measurement, lowest intensity was represented as single + sign and each fold increase was denoted by additional + sign. Number of positive cells as well as intensity of H-2K^k was significantly higher in the rvv-IL-2 vaccinated group (179±11, +++, P< .0001)) compared to the control rvv-lacZ (45±8, +) or PBS group (36±6, +). Expression of VEGF-R was significantly lower in the rvv-IL-2 vaccinated group (+) compared to the control rvv-lacZ (+++) or PBS (+++) group. Thin arrow (®) indicated positive staining where as thick arrow (è) indicated negatively stained cells in case of H-2K^k expression.

cells in the control rvv-lacZ or PBS group did not express MHC class I protein (thick arrows). In addition, the expression of MHC class I protein was also low in the rvv-lacZ or PBS vaccinated group (+) compared to the rvv-IL-2 treated group (+++). It appeared that treatment with rvv-lacZ vaccine restored MHC class I to some extent, however, treatment with rvv-IL-2 vaccine significantly restored class I expression.

VEGF stimulates the proliferation of endothelial cells through its specific receptor, such as flk-1 or flt-1 (Strawn et al, 1996) and induces formation of new blood vessels or angiogenesis. Results in Table 1 demonstrated that tumor homogenates from PBS treated mice secreted VEGF, which is inhibited by treatment with rvv-IL-2 vaccine. To determine whether number of VEGF receptors (VEGF-R) is also increased in tumor tissues, we performed immunohistochemistry using anti-flk-1 antibody. Results presented in Figure 4 show that the tumor associated endothelial cells adjacent to the vessels stained strongly (+++) for flk-1 protein in the PBS and rvv-lacZ treated groups. The extent of staining of the tumor sections by flk-1 antibody from mice treated with rvv-IL-2 was considerably reduced (+). The number of positive foci was also higher in the control vaccinated groups compared to the rvv-IL-2 vaccinated group. These results suggested that microenvironment of SCC VII/SF tumors favors angiogenesis. Treatment with subcutaneous injection of irradiated, rvv-IL-2 infected SCCV II/SF tumor cells resulted in inhibition of angiogenesis.

E. Role of rvv in subcutaneous vaccination

Although the intratumoral growth of rvv was not affected by repeated intratumoral injections, subcutaneous injection of rvv may induce anti-vaccinia immunity (Schlom et al, 2003) and eventually neutralize the rvv vaccines. To overcome this potential problem we tested whether we could avoid the use of rvv for subcutaneous immunization. We used a combination of irradiated SCC VII/SF cells mixed with either 125 units of rhIL-2 or recombinant murine GM-CSF for the subcutaneous injection without infecting the cells with rvv. Helper T cell activities by T-cell proliferation assays of immune cells from mice vaccinated once with these vaccines were compared with those with irradiated, rvv-infected SCC VII/SF cells. Results of these experiments are summarized in Figure 5. Helper activities of immune cells from mice vaccinated with subcutaneous injection with irradiated SCC VII/SF cells mixed with rhIL-2 were significantly higher in splenocytes as well as TDLN cells compared to irradiated tumor cells alone (P< .02). However, these activities were lower compared to those with rvv-IL-2 vaccines (P< .004). More accurate tumor specific helper activities are reflected by the amounts and nature of cytokines secreted by the immune cells after stimulation with the tumor cells. We determined the levels of Th1 and Th2 cytokines secreted by the splenocytes and TDLN cells from vaccinated mice and the results are presented in Table 3. Levels of Th1 cytokines secreted by splenocytes are similar in groups of mice vaccinated with rvv-IL-2 infected tumor cells and the groups vaccinated with tumor cells mixed with rhIL-2. Amounts of Th1 cytokines secreted by TDLN cells were, however, slightly lower in groups of mice vaccinated with tumor cells mixed with rhIL-2 compared to those vaccinated with tumor cells infected with rvv-IL-2. The levels of Th2 cytokines were, however, higher in rhIL-2 groups both in Spleen and TDLN cells, similar to levels with irradiated SCC VII/SF cells alone. Th1 cytokines secreted by immune cells from mice vaccinated with tumor cells mixed with rGM-CSF were lower than the levels obtained with mice treated with rvv-IL-2 infected tumor cells. Levels of Th2 cytokines were similar in groups of mice treated with irradiated SCC VII/SF cells and those treated with rvv-IL-2 infected cells.

Cytotoxic T cell activities of immune cells from mice vaccinated with tumor cells infected with rvv-IL-2 and those vaccinated with tumor cells mixed with either rhIL-2 or rGM-CSF were also compared by Chromium release assays. Results in Figure 6 demonstrate that significant cytolytic activities of immune cells were found when mice were vaccinated with rhIL-2 mixed with tumor cells compared to irradiated tumor cells alone (P<. 02). However, these activities were lower compared to immune cells from mice vaccinated with rvv-IL-2 infected tumor cells (P<. 01). No differences in CTL activities of immune cells from mice vaccinated with irradiated SCC VII/SF cells mixed with rGM-CSF and immune cells from those vaccinated with irradiated tumor cells alone were found.

Figure 5. Tumor specific helper T cell activities induced by single subcutaneous vaccination. T cell proliferation assay was performed and stimulation index (S.I.) of bulk splenocytes (A) and TDLN cells (B) was determined as described in Materials and Methods. Data represent Mean±SE value of triplicate well. Vaccination was administered on day 7 after tumor implantation. Bulk splenocytes or TDLN cells from 4 mice of each vaccinated group was harvested on day 9, pooled and stimulated for 5 days in a tissue culture incubator using irradiated SCCVII/SF or C15 tumor cells as stimulants. Concavalin A was used as positive control stimulant whereas culture medium served as the background stimulant. Stimulation indices of bulk splenocytes (A) or TDLN cells (B) was significantly higher in the group vaccinated with irradiated SCC cells plus rhIL-2 compared to the groups vaccinated with irradiated SCC cells alone, irradiated SCC cells plus GM-CSF, rvv-lacZ or PBS (P< .02). Higher stimulation index was achieved by rvv-IL-2 vaccination in spleen (A) as well as TDLN (B) compared to all the other groups (P< .004). Stimulation in the presence of antigen negative C15 cells was low compared to the SCCVII/SF cells.

Table 3. Secretion of cytokines by immune cells following subcutaneous injection with irradiated tumor cells plus recombinant IL-2 or GM-CSF

__________________________________________________________________________________

Cytokines^a

__________________________________________________________________________________________________

Source of IL-2 IFN-g IL-4 IL-5 IL-10

Immune Groups

Cells

__________________________________________________________________________________________________

Spleen rvv-IL-2 177±7 1567±57 34±3 19±2 29±7

Spleen SCC 86±5 130±51 99±30 39±3 129±15

Spleen IL-2 122±12 1762±52 70±8 36±2 110±17

Spleen GM-CSF 107±18 774±54 89±8 32±3 104±15

TDLN rvv-IL-2 120±10 30±1 17±2 45±3 23±9

TDLN SCC 69±8 11±9 41±13 56±6 72±5

TDLN IL-2 105±7 19±1 42±11 53±12 56±9

TDLN GM-CSF 73±4 19±.5 45±12 46±5 53±6

__________________________________________________________________________________________________

^aAmounts of cytokines (pg/ml±SE) secreted by the immune cells were determined using ELISA kit. Immune cells were isolated from spleen and TDLN as described in Materials and Methods. Mice were vaccinated on day 7 with irradiated SCCVII/SF cells (SCC), irradiated SCCVII/SF cells infected with rvv-IL-2 or mixed with 125 units of either IL-2 or GM-CSF as described in Materials and methods.

Figure 6. Induction of cytolytic activities of immune cells from spleen and TDLN by single subcutaneous vaccination. Mice were vaccinated on day 7 after tumor implantation. Cytolytic activities were assayed on splenocytes (A) and TDLN cells (B) of vaccinated mice on day 9. The immune cells were harvested on day 9 from 4 mice of each group, pooled and cultured in the presence of irradiated SCCVII cells and rhIL-2 for 7 days as described in Materials and Methods. [⁵¹Cr] labeled SCCVII/SF cells were used as target in the cytolytic assay. Cytolytic activities of bulk splenocytes (A) or TDLN cells (B) was significantly higher in the group vaccinated with irradiated SCC cells plus rhIL-2 compared to the groups vaccinated with irradiated SCC cells alone, irradiated SCC cells plus GM-CSF, SCC plus rvv-lacZ or PBS (P< .02). Cytolytic activities were higher in the rvv-IL-2 vaccinated group in spleen (A) as well as TDLN (B) compared to all the other groups (P< .01). Cytolytic activities were low and negligible in the groups vaccinated with irradiated SCC cells alone, SCC cells plus GM-CSF, SCC plus rvv-lacZ or PBS.

IV. Discussion

Intratumoral vaccination of mice bearing SCC VII/SF tumor cells in the floor of the mouth with rvv-IL-2 inhibited growth of tumors and enhanced survival, although tumor regression was not observed (Qin et al., 2001). We also found that the immune cells from tumor bearing mice only partially responded to mitogens. Comparison of the expression of a number of IMM molecules between the cultured tumor cells and tumor homogenate showed higher expression of IMM in tumor homogenate. We hypothesized that the failure to induce tumor regression was due to the synthesis of tumor-induced IMM in the tumor microenvironment (Qin et al, 2001). Removal of the IMM by specific antibodies or inhibition of their synthesis by anti-sense oligonucleotides could make the vaccine more effective. However, the vaccination protocol for patients will become extremely complicated. Subsequently we found that immunization with irradiated, rvv-IL-2 infected tumor cells combined with intratumoral injection of rvv-IL-2 induced tumor regression and induction of cellular and helper T cell activities (Dasgupta et al, 2003), suggesting the reversal of immune suppression.

In this study we compared the levels of a number of IMM in tumor homogenate from 3 groups of mice vaccinated with (1) PBS, (2) by a single subcutaneous injection of irradiated SCC VII/SF cells, infected with rvv-IL-2 as well as (3) the control vaccine rvv-lacZ. The results demonstrated that the vaccination with rvv-IL-2 reversed the expression of IMM in the tumor microenvironment. We also investigated the possible mechanism of IMM action in this murine model.

GM-CSF has been found to be most potent in generating systemic anti-tumor responses (Danna et al, 2004). GM-CSF has multiple functions in immune regulation. Among these functions, GM-CSF has been shown to augment antigen presentation in a variety of cells (Morrissey et al, 1987), enhance major histocompatibility complex class II antigen expression on monocytes (Blanchard and Djeu, 1991), increase the expression of adhesion molecules on granulocytes and monocytes (Arnaout et al, 1986) and participate in the amplification of T-cell proliferation (Santoli et al, 1988). GM-CSF also induces the differentiation and maturation of hematopoietic cells (Metcalf, 1985). Tumor derived GM-CSF, on the other hand, induces the proliferation of a population of CD34+ cells, which suppress the functions of intratumoral T cells. Patients whose HNSCC contain a higher number of CD34+ cells have increased incidence of subsequent recurrence and reduced survival (Young et al, 1996a; Young et al, 1997). Murine studies demonstrated that expression of GM-CSF by progressive tumors is associated with increased metastasis (Takeda et al, 1991; Young et al, 1996b). Tumor homogenates from PBS treated mice expressed significant amounts of GM-CSF, both at the mRNA and protein levels. Following subcutaneous injection with irradiated, rvv-IL-2 infected tumor cells expression of GM-CSF was reduced by about 80% both at the mRNA (Figure 1) and protein (Table 1) levels. Injection of the control rvv-lacZ vaccine only reduced the GM-CSF mRNA by 34% (Figure 1) and GM-CSF protein was marginally stimulated by this treatment (Table 1).

IL-10 is a potent inhibitory factor for dendritic cells cell functions. Addition of IL-10 to splenic DC impairs their capacity to produce IL-12 and induce a Th1 response in vivo (De Smedt et al, 1997). In human, IL-10 has been shown to inhibit the ability of DC to stimulate T cells by preventing the induction of co-stimulatory molecules CD86 (Vicari et al, 2002). Ovarian cancer biopsies express higher levels of IL-10 mRNA compared to normal ovaries (Pisa et al, 1992). By subcutaneous injection of irradiated tumor cell, infected with rvv-IL-2 levels of IL-10 and the levels of mRNA encoding IL-10 were reduced by over 90%. Reduction of IL-10 by vaccination with the control rvv-lacZ vaccine was about 30% both at the mRNA and protein levels (Figure 1, Table 1).

TGF-b1 also has been shown to suppress the maturation and functions of DC (Albini et al, 1987; Young et al, 1996b; Doran et al, 1997). Reduction of TGF-b1 by rvv-IL-2 vaccination was less than 20% at the mRNA level, although subcutaneous vaccination with rvv-IL-2 infected tumor cells resulted in about 70% reduction of the levels of TGF-b1 protein in the tumor homogenate (Figure 1, Table 1). iNOS also suppresses DC maturation (Young et al, 1996b). Significant reduction of iNOS resulted by the rvv-IL-2 vaccination both at the mRNA (Figure 1) and protein level (Figure 2). VEGF also suppresses the maturation and functions of DC (Oyama et al, 1998; Saito et al, 1998). Vaccination with rvv-IL-2 resulted in the reduction of the expression of mRNA encoding VEGF by about 40% and significantly inhibited the expression of VEGF protein by 90% (Figure 1, Table 1).

Reduction of the levels of IMM in the tumor microenvironment of mice treated with subcutaneous injection of tumor cells infected with rvv-IL-2 resulted in the induction of both helper and cytotoxic T cells (Table 2, Table 3, Figure 5 and Figure 6). Helper T cells play a critical role in CD8+ T cell mediated anti-tumor immunity (Keene and Forman, 1982). Helper T cells have two subtypes, Th1 and Th2, which can be identified from the profile of cytokine secreted by the helper cells in response to the stimulation by the antigen. Th1 cells secrete IL-2 and IFN-g, while Th2 cells secrete IL-4, IL5 and IL-10 (Swain, 1995; Abbas et al, 1996). Th1 cells are associated with induction of CTL responses, whereas Th2 cells are involved in promoting antibody responses. Cytokines produced by each cell subtype are antagonistic for the proliferation of the other subtype. As a result Th2 cells could negatively impact CTL generation, which is likely to reduce the efficacy of cancer immunotherapy. Results presented in Table 2 demonstrated that helper T cells generated by rvv-IL-2 vaccination are Th1 type. However, subtype of T helper cells generated by the control vaccine, rvv-lacZ is unbiased.

For the activation of T cells to acquire anti-tumor immunity, tumor antigens need to be presented by antigen presenting cells (APC) to naïve helper and cytotoxic T cells. SCC VII/SF tumor cells cannot act as APC since these cells do not express MHC class II or B7 co-stimulatory molecules (Qin et al, 2001). Therefore, induction of tumor specific T cell activation by the rvv-IL-2 vaccine likely involved other types of APC, such as DC. Immunohistochemistry of tumor sections from mice treated with rvv-IL-2 and control vaccines showed only a few DC in the tumor bed from mice vaccinated with the control vaccines and numerous mature DC in the tumor beds from mice vaccinated with rvv-IL-2. Numbers of both CD4+ and CD8+ T cells were also high in parallel sections from rvv-IL-2 treated mice (Figure 3). Proximity of DC and the T cells in the tumor beds is likely to facilitate antigen presentation and induction of tumor specific immunity. Decreased number of PCNA positive tumor cells in the tumor beds of rvv-IL-2 mice suggests inhibition of tumor cell proliferation induced by the vaccine.

Cytotoxic T cells recognize antigenic peptides from tumor antigens, which are bound in the cleft of MHC class I molecules. If tumor antigens are not presented bound to MHC class I molecule, tumor cells will not be recognized by the activated T cells. Thus, total or partial loss of MHC class I molecule is a mechanism of tumor immune escape (Garrido et al, 1993). MHC class I loss has been reported to occur frequently in human malignancies, including HNSCC (Grandis et al, 1995). Cultured SCC VII/SF cells express MHC class I molecules. However, fresh tumors from tumor bearing mice show low expression of MHC class I molecule (Figure 4). IL-10 has been reported to down regulate MHC class I molecule in tumor cells (Salazar-Onfray et al, 1997; Yue et al, 1997; Zeidler et al, 1997). Expression of IL-10 and the reversal by rvv-IL-2 vaccination also resulted in restoration of MHC class I molecule (Figure 4). Thus, the enhanced CTL activities by rvv-IL-2 vaccination may be also due to restoration of MHC class I expression. Although rvv-IL-2 vaccination appeared to result in the restoration MHC class I expression by the tumor cells in vivo, the exact mechanism is yet not clear. One possibility is the secretion of INF-g by the immune cells generated by the rvv-IL-2 vaccine.

VEGF stimulates the proliferation of endothelial cells through its specific receptor, such as flk-1 or flt-1 (Strawn et al, 1996) and induces formation of new blood vessels or angiogenesis. Angiogenesis is required for solid tumor growth (Folkman, 1971, 1990) and facilitates tumor progression and metastasis (Weidner et al, 1991). Results in Table 1 demonstrated that tumor homogenates from PBS treated mice secreted VEGF, which is inhibited by treatment with rvv-IL-2 vaccine. To determine whether number of VEGF receptor was also increased in tumor tissues, we performed immunohistochemistry using anti-flk-1 antibody. Results presented in Figure 4 showed that the tumor associated endothelial cells adjacent to the vessels stained strongly for flk-1 protein. The extent of staining of the tumor sections by flk-1 antibody from mice treated with rvv-IL-2 was considerably reduced, as was the number of positive foci. These results suggested that microenvironment of SCC VII/SF tumors favors angiogenesis, which may lead to tumor progression and metastasis. Treatment with subcutaneous injection of irradiated, rvv-IL-2 infected SCCVII/SF tumor cells resulted in inhibition of angiogenesis, thereby preventing tumor progression and metastasis.

For the subcutaneous vaccine to be effective the tumor cells were infected with rvv-IL-2 and rvv-lacZ had marginal effect. These suggested that the reversal of immune suppression was not the result of vaccinia virus. Most likely, the reversal of immune suppression was the result of the generation of IL-2 from rvv-IL-2. Although the growth of rvv in the tumors was not affected by repeated intratumoral injections of rvv-IL-2, subcutaneous injection of rvv may induce anti-vaccinia immunity (Schlom et al, 2003). To overcome this potential problem we tested whether we could avoid the use of rvv for subcutaneous immunization. We used rhIL-2 and murine rGM-CSF mixed with irradiated SCC VII/SF cells for subcutaneous vaccination. IL-2 has the potential to expand tumor specific T cells, while GM-CSF has been reported to enhance local recruitment of DC to the vaccine site and thereby increase antigen presentation. Results presented in Figure 5, Figure 6 and Table 3 demonstrate that while rGM-CSF had no effect, rhIL-2 stimulated both the helper and CTL activities, although rhIL-2 was less effective compared to rvv-IL-2. Helper T cell activities induced by rhIL-2 determined by T cell proliferation were higher compared to those of irradiated SCC VII/SF cells (Figure 5), although the differences were not statistically significant. However, the results were similar when the experiments were repeated one more time. Moreover, amounts of Th1 cytokine secretion from immune cells of mice vaccinated with rhIL-2 were similar to levels from mice treated with rvv-IL-2 (Table 3). In these experiments, we used only one concentration of cytokine, 125 Units/injection. Since the effects of these cytokines are strictly concentration dependent (Serafini et al, 2004), we need to explore various concentrations of these cytokines for vaccine preparations.

One problem with the use of irradiated tumor cells in the subcutaneous vaccination is that the vaccine becomes more complicated and may become patient dependent. We have identified a number of proteins, which were highly expressed in tumor tissues and had low expression in normal organs. These proteins can be considered as tumor-associated antigens (unpublished data). In future studies we will explore the use these proteins for subcutaneous immunization, rather than the whole tumor cells.

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Sunil K. Chatterjee