T-regulatory cells are relatively deficient in squamous ...

Cancer Gene Therapy (2007) 14, 573–582 r

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ORIGINAL ARTICLE

T-regulatory cells are relatively deficient in squamous carcinomas undergoing regression in mice immunized with a squamous carcinoma vaccine enriched for immunotherapeutic cells A Chopra1, I O-Sullivan1, J Carr1, TS Kim2 and EP Cohen1 1

Department of Microbiology and Immunology, University of Illinois College of Medicine, Chicago, IL, USA and 2School of Life Sciences and Biotechnology, Korea University, Seoul, Korea In a prior report (Int J Cancer 2006; 119: 339–348), we described a new vaccination strategy for squamous cell carcinoma (SCC). The vaccine was prepared by transfer of unfractionated DNA-fragments (25 kb) from KLN205 cells, a squamous carcinoma cell line (DBA/2 origin; H-2d) into LM cells, a highly immunogenic mouse fibroblast cell line (C3H/He origin; (H-2k)). As only a small proportion of the transfected cell population was expected to have incorporated DNA segments that included genes specifying antigens associated with the squamous carcinoma cells, we devised a novel strategy to enrich the vaccine for immunotherapeutic cells. Enhanced immunity to squamous carcinoma was induced in tumor-bearing mice treated solely by immunization with the enriched vaccine, which translated into prolonged survival without toxicity. Here, we describe the characteristics of the cell populations infiltrating established squamous carcinomas undergoing regression in mice immunized with vaccines enriched for immunotherapeutic cells. The results indicated that CD8 þ T cells were predominant and that T-regulatory cells (FoxP3 þ , CD4/ CD25 þ , CD4/CD62Lhigh, CD4/CTLA-4e) were relatively deficient in the regressing tumors. Inflammatory infiltrates were not detected in various organs and tissues of mice immunized with the DNA-based vaccine. Cancer Gene Therapy (2007) 14, 573–582. doi:10.1038/sj.cgt.7701040; published online 23 March 2007 Keywords: DNA-based vaccine; immunotherapy; mice; squamous carcinoma; T-regulatory cells

Introduction

Although experimental protocols in mice are revealing the potential of tumor immunotherapy, effective vaccination strategies in cancer patients are less successful. One possible explanation is that activated T-regulatory (Treg) cells, which predominate in the tumor, inhibit cellular antitumor immune responses induced by the vaccine. This is of special concern in squamous carcinoma. Squamous carcinomas cells (SCC) are notoriously immunosuppressive.1–5 A second possible explanation is that many cellular vaccines used for cancer therapy do not contain a sufficient number of immunotherapeutic cells, that is, cells that are responsible for inducing the antitumor immune response. As the vast majority of structures expressed by cancer cells are normal cellular constituents, rather than ‘tumor antigens’, it is likely that relatively few cells in vaccines prepared by transfer of unfractionated Correspondence: Dr EP Cohen, Department of Microbiology and Immunology (m/c 790), 835 South Wolcott Ave, Chicago, IL 60612, USA. E-mail: [email protected] Received 7 August 2006; revised 13 December 2006; accepted 11 February 2007; published online 23 March 2007

tumor-derived peptides,6,7 tumor-cell lysates,8,9 mRNA,10,11 cDNA,12 apoptotic-cell bodies13,14 or heat shock proteins15,16 into syngeneic dendritic cells (DC) specify TAA. In prior reports,17–20 we described the results of studies in tumor-bearing mice including mice with SCC treated by immunization with a unique vaccine prepared by transfer of sheared genomic tumor-DNA-fragments into LM cells, a mouse fibroblast cell line. As the transferred DNA integrates spontaneously into the genome of the recipient cells, replicates as the cells divide and is expressed, the number of vaccine cells could be conveniently expanded as desired for multiple rounds of immunization. Sufficient DNA to prepare the vaccine was obtained from tumors as small as 4 mm, enabling treatment at an early stage of the disease. The fibroblast cell line selected as the recipient of DNA from the tumor expressed allogeneic MHC class I-determinants. Allogeneic MHC-determinants, which are strong immune adjuvants,21–23 stimulate uptake of the vaccine by DC of the host where TAA are expressed (cross priming).24,25 They ensure that the vaccine, as other foreign tissue grafts, will be rejected. However, like other cellular tumor vaccines, relatively few cells in the DNA-based vaccine were expected to have

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incorporated DNA-fragments that included genes that specified TAA. To address this issue and to increase the therapeutic properties of the vaccine, we devised a novel strategy to enrich the vaccine for immunotherapeutic cells. The enrichment strategy involved dividing the population of transfected cells into a number of small pools, expanding the number of cells derived from the individual pools, identifying pools that stimulated immunity to the squamous carcinoma to the greatest extent, followed by successive rounds of positive immune-selection. DBA/2 mice with established tumors derived from KLN205 cells, a highly malignant squamous carcinoma cell line isolated originally from the Nettesheim lung carcinoma, treated by immunization with vaccines enriched for immunotherapeutic cells survived significantly longer than mice in various control groups, including mice immunized with transfected cells from non-enriched populations.18 Here, we describe the characteristics of the cell populations infiltrating established squamous carcinomas undergoing regression in mice immunized with vaccines enriched for immunotherapeutic cells. The results in tumor-bearing mice treated successfully by immunization with the enriched vaccines revealed a predominance of CD8 þ T cells and a relative deficiency of T-reg cells in the regressing tumors.

Materials and methods

Experimental animals, tumor cell lines and monoclonal antibodies Eight- to 10-week-old pathogen-free DBA/2 female mice (H-2d) were from the Jackson Laboratory (Bar Harbor, ME). The animals, between 10 and 14 weeks old when used in the experiments, were maintained according to NIH Guidelines for the Care and Use of Laboratory Animals. KLN205 cells were from the American Type Culture Collection (ATCC). LM cells, a fibroblast cell line of C3H/He mouse origin, were also from the ATCC. KLN205 cells were maintained by serial passage in histocompatible DBA/2 mice, or at 371 in a humidified 7% CO2/air atmosphere in Dulbecco’s modified Eagle medium (Gibco BRL, Grand Island, NY) supplemented with 10% heat-inactivated fetal bovine serum (Sigma, St Louis, MO) and antibiotics (Gibco BRL) (growth medium). LM cells were maintained in growth medium under the same conditions. Monoclonal antibodies (mAbs) for determinants associated with CD8 þ , CD4 þ and NK1.1 cells were from Pharmingen, (San Diego, CA). mAbs for CD25, CD62L, CTLA-4e-determinants were from B-D Biosciences, (San Jose, CA). Low tox rabbit complement (C) was from Pel Freeze, (Rogers, AR). Primers for FoxP3 were from Sigma, (St Louis, MO). Recovery of genomic DNA from KLN205 cells A DNeasy isolation kit (Qiagen, Valencia, CA) was used to obtain genomic DNA from KLN205 cells, according to the manufacturer’s instructions, as described previously.18

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The A260/A280 ratio of the isolated DNA was 41.8 in each instance. The molecular size of the extracted DNA was approximately 25 kb, as determined by agarose gel electrophoresis.

Modification of LM fibroblasts to secrete IL-2 As a means of augmenting their nonspecific immunogenic properties, the fibroblasts were modified by transduction with a replication-defective retroviral vector (pZipNeoSVIL-2) to secrete interleukin 2 (IL-2) before transfection with DNA from KLN205 cells (LM-IL-2), as described previously.18 An analysis by enzyme-linked immunosorbent assay of the culture supernatants of LM-IL-2 cells indicated that 106 transduced cells formed 196 pg IL-2/ml/106 cells/48 h. Immunofluorescent staining and cytofluorometric measurements Quantitative immunofluorescence measurements were used to measure the expression of MHC class I/IIdeterminants and co-stimulatory molecules by the fibroblasts used as recipients of DNA from KLN205 cells, as described previously.18 One-parameter fluorescence histograms were generated by analyzing at least 1 104 cells in each instance. Modification of the cytokine-secreting fibroblasts to express allogeneic H-2Kb-class I-determinants Allogeneic class I-determinants are strong immune adjuvants.27–29 To further stimulate uptake of the vaccine by DC of DBA/2 mice, the fibroblasts (H-2k) were modified to express additional allogeneic MHC class I-determinants (H-2Kb), as described previously.18 Preparation of the DNA-based cellular vaccine The vaccine was prepared by transfer of sheared unfractionated DNA-fragments from KLN205 cells into LM fibroblasts, modified beforehand to secrete IL-2 and to express H-2Kb-determinants (LM-IL-2Kb cells). The method described by Wigler et al.26 was used, modified as described previously.17,18 Mouse interferon-g ELISPOT assays Mouse ELISPOT interferon-g (IFN-g) assays were used to detect the presence of spleen cells responsive to KLN205 cells in DBA/2 mice immunized with the modified, transfected fibroblasts, as described previously.18 The spots were counted by computer-assisted image analysis (ImmunoSpot Series 2 analyzer: Cellular Technology Limited, Cleveland, OH). Detection of cytotoxic T lymphocytesreactive with KLN205 cells by 51Cr-release cytotoxicity assays Standard 51Cr-release cytotoxicity assays were used to detect the presence of spleen cells with cytotoxic activity toward KLN205 cells in mice immunized with the transfected fibroblasts, as described previously.18 The quantity of isotope released was measured in a gamma counter (Beckman, Palo Alto, CA).


The percent specific cytolysis was calculated as: Experimental 51 Cr release Spontaneous 51 Cr release 100 Maximum 51 Cr release Spontaneous 51 Cr release

The spontaneous release of release in each instance.

51

Cr was o15% of the total

Strategy for the enrichment of the cellular vaccine for cells that induce immunity to KLN205 cells in DBA/2 mice Identification of highly immunogenic (immunohigh) pools of transfected cells. As only a small proportion of cells in the vaccine was expected to incorporate DNA fragments that included genes responsible for induction of the antitumor immune response, a novel strategy was employed to enrich the vaccine for immunotherapeutic cells, as described previously.18 In brief, small aliquots (1 103) of the suspension of transfected cells were added to 10 individual wells of a 96-well plate. We reasoned that if the starting inoculums were sufficiently small, then some pools would contain greater numbers of immunotherapeutic cells than others. Pools containing greater numbers of immunotherapeutic cells could be identified by their heightened immunogenic properties against KLN205 cells in DBA/2 mice. As the cell number increased, cells from individual pools were transferred to progressively larger cell culture plates, and then flasks. After the number of cells from individual wells had increased to about 5 107, a portion of the expanded cell population from each pool was collected and maintained frozen/viable (for later recovery). The remaining portion was used to immunize naı¨ ve DBA/2 mice. After immunization, two independent means (ELISPOT-IFN-g and 51Cr-release cytotoxicity assays) were used to identify pools that stimulated spleen cellmediated immunity toward KLN205 cells to the greatest (immunohigh) and least (immunolow) extent. Frozen/viable cells from each of these pools were recovered and the procedure was repeated for two additional rounds of either positive or negative immune selection, using 1 103 transfected cells as the starting inoculums in each instance. The immunogenic properties of transfected cells from subpool (sp) 6-10-1 after three rounds (31) of positive selection exceeded those of any of the other pools (sp 6-10-1 ¼ immunohigh). In a similar manner, cells from sp 9-6-2 after three rounds (31) of negative selection stimulated immunity to KLN205 cells to the least extent (sp 9-6-2 ¼ immunolow). RT-PCR for FoxP3 Reverse transcription-polymerase chain reaction (RTPCR) was used to detect the presence of FoxP3 in tumors undergoing regression in mice immunized with the DNA-based vaccines. RNA was isolated using RNeasy Kits from Qiagen (Valencia, CA), according to the manufacturer’s instructions. Approximately 6 106 cells were disrupted by addition of 600 ml of lysis buffer (RLT) with 1 v/v% b-mercaptoethanol. The cells were homogenized and 1 volume of 70% ethanol was added before the extracts were loaded onto RNeasy mini columns. The

columns were washed and RNA was eluted with 30 ml of RNase-free water. RT-PCR was performed with a one-step RT-PCR kit from Qiagen (Valencia, CA), according to the manufacturer’s instructions. In brief, 10 ng RNA was mixed with buffer containing 1.25 mM MgCl2, 40 mM dNTPs, 0.6 mM each forward and backward primers and 2 ml enzyme mixture containing Reverse Transcriptase and Taq Polymerase. The reverse transcriptase reaction was performed at 501C for 45 min. The PCR reaction was at 941C denaturation step for 2 min, 571 annealing step for 1 min and 721C extension for 2 min for 35 cycles using a DNA Thermal Cycler 480 (Perkin-Elmer, Wellesley, MA). The primers used for the analysis of the FoxP3 gene were as follows: forward 50 -CAGCTGCCTACAGTGCCCCTA, backward 50 -CATTTGCCAGCAGTCGGTAG.

Statistical analyses Kaplan–Meier log rank analyses were used to determine the statistical differences between the survival of mice in the various experimental and control groups. Po0.05 was considered significant. Student’s t-test one-way analysis of variance was used to determine the statistical differences between experimental and control groups in the experiments performed in vitro.

Results

Enhanced immunity to squamous carcinoma in DBA/2 mice immunized with a squamous carcinoma vaccine enriched for immunotherapeutic cells A unique vaccine for KLN205 cells, a highly aggressive squamous carcinoma cell line of DBA/2 mouse origin (H-2d), was prepared by transfer of DNA-fragments from the squamous carcinoma cells into LM cells, a mouse fibroblast cell line (C3H/He mouse origin, (H-2k)). As relatively few cells in the vaccine were expected to have incorporated DNA fragments that included genes that specified TAA, a novel enrichment strategy was used to increase the number of immunotherapeutic cells. As indicated (Figure 1a), after three rounds of positive immune selection, the proportion of cells in the vaccine that stimulated cytotoxic T lymphocytes (CTL) responses toward the SCC increased approximately 18-fold, from 1 in 900 000 in the non-enriched population to 1 in 50 000 in the enriched population. As a further means of investigating the relative immunotherapeutic properties of the enriched and nonenriched vaccines, the number of cells in the enriched population required to stimulate one-half the maximal T-cell response toward KLN205 cells in spleen cells from DBA/2 mice was compared with the number of cells in the non-enriched cell population required to stimulate an equivalent response. ELISPOT IFN-g assays were used to compare the relative immunogenic properties of cells from the immunohigh pool (31), the immunolow pool (31) and the non-enriched master pool. Naı¨ ve DBA/2 mice received two subcutaneous (s.c.) injections at weekly intervals of 4 106 cells from each of the cell pools. One group of

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Figure 1 The enrichment strategy increased in the number of immunotherapeutic cells in the vaccine. (a) An assay based on limiting dilution and the application of Poisson statistics was used to determine the number of cells in the immunohigh pool (31) that stimulated CTL responses toward KLN205 cells. Varying numbers (range 1 103 to 5 105) of (mitomycin C-treated) cells from the immunohigh pool (31) or, for comparison, from the non-enriched master pool (LM-IL-2Kb/KLN) were distributed to 12 replicate wells at each cell number, followed by the addition of spleen cells from DBA/2 mice. The mice had been injected s.c. two times at weekly intervals with 4 106 cells from the immunohigh pool (31) or the nonenriched master pool beforehand (ratio spleen cells: vaccine cells ¼ 10:1). The cell culture plates were incubated for 5 days under standard cell culture conditions, to allow the cells to increase in number. (Media changes were performed as required.) Afterward, 51Cr-labeled KLN205 cells were added and a specific isotope release assay was performed. (b) Cells (4 106) from the immunohigh pool (31), the immunohigh pool (2o), the immunolow pool (31) or the non-enriched master pool (LM-IL-2Kb/KLN) were injected s.c. two times at weekly intervals into the left flank of naı¨ve DBA/2 mice. One week after the second injection, spleen cells from the immunized mice were serially diluted twofold, followed by the addition of KLN205 cells (ratio spleen cells: KLN205 cells ¼ 10:1). After 18 h further incubation, an ELISPOT IFN-g assay for the presence of spleen cells responsive to KLN205 cells was performed.

mice was not immunized. One week after the second injection, spleen cells from the immunized mice were serially diluted twofold and co-cultured for 18 h with KLN205 cells. At the end of the incubation, ELISPOT IFN-g assays were performed. As indicated (Figure 1b), the number of spleen cells from mice immunized with cells from the immunohigh pool (31) required to reach the half maximal response was significantly (Po0.001) less than number of spleen cells from mice immunized with cells from the immunolow pool (31) or mice immunized with cells from the non-enriched master pool. Intermediate values were obtained if the spleen cells were from mice immunized with cells from the immunohigh pool (21). Thus, as determined by two independent assays, the enrichment strategy resulted in an increase in the number of cells in the vaccine that stimulated immunity to the SCC.

T-reg cells were relatively deficient in squamous carcinomas in mice treated by immunization with vaccines enriched for immunotherapeutic cells T-regs are a unique class of cells derived from the thymus. They are critical in the regulation of self-reactive T cells. The cells, which represent about 10% of CD4 þ cells, express the a-chain of the IL-2-receptor in high density (CD25high). They prevent the development of self-reactive T cells, as indicated by the finding that the transfer of CD4 þ CD25high cells to immunodeficient mice injected with CD4 þ CD25 () cells inhibited the development of autoimmune disease.27–29

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Classic studies indicate that T-reg cells can promote tumor growth by suppression of T-cell-mediated antitumor immunity.30–32 This is especially the case in squamous carcinoma. Squamous carcinomas are notoriously immunosuppressive. To determine if the heightened antitumor immune responses in tumorbearing mice immunized with cells from the immunohigh pool (31) affected the proportion of tumor infiltrating T-reg cells, the percentage of T-reg cells in established SCC in mice immunized with cells from the immunohigh pool (31) was compared with the percentage of tumor infiltrating T-reg cells in mice immunized with cells from the immunolow pool (31). Naı¨ ve DBA/2 mice received an s.c. injection of 1 106 KLN205 cells into the left flank. Six days later, when the tumors were 5 to 8 mm in diameter, the mice received a single s.c. injection of 4 106 cells from the immunohigh pool (31) or the immunolow pool (31) into the right flank. Eleven days afterward, cells from tumors in the immunized mice were analyzed by immunofluorescent staining for the presence of CD4/CD25 þ , CD4/CD62Lhigh and CD4/ CTLA-4e-determinants, characteristic of T-reg cells. As additional controls, the same procedure was followed except that the tumor-bearing mice were immunized with equivalent numbers of cells from the non-enriched master pool, or the tumor-bearing mice were not injected with the vaccine. As indicated (Figure 2), the proportion of CD25 þ , CD62L þ and CTLA-4 þ cells in tumors from mice immunized with cells from the immunohigh pool (31) was significantly less (Po0.01) than in tumors from mice


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Figure 2 T-regulatory cells were relatively deficient in squamous carcinomas in mice immunized with cells from the immunohigh pool (30). KLN205 cells (1 106) were injected s.c. in the left flank of DBA/2 mice. Six days later, 4 106 cells from the immunohigh (31) pool were injected s.c. in the right flank of the tumor-bearing mice. Eleven days afterward, the mice were euthanized, the tumors were removed and pooled cell suspensions from two mice were prepared by incubation in HBSS containing a mixture of collagenase, hyaluronidase and DNase (Sigma). The tumor cell suspension was stained with PE-Cy5-labeled CD3 FITC-labeled CD4/PE-labeled CD25 mAbs, with FITC-labeled CD4/PE-labeled CD62L mAbs or FITC-labeled CD4/PE-labeled CTLA-4e mAbs, followed by analysis in a FACS. As controls, the same procedure was followed except that cells from the immunolow (31) or the non-enriched master pool (LM-IL-2Kb/KLN) were substituted for cells from the immunohigh (31) pool, or the cells were from mice injected with KLN205 cells alone. A minimum of 20 000 live cell events gated by forward and side scatter patterns, followed by gating on CD3 cells, were analyzed. The numbers indicate the percentage cells in the specific quadrants.

immunized with cells from the immunolow pool (31). The highest proportion of T-reg cells was in tumors from untreated mice injected with KLN205 cells alone.

Differences in the proportion of CD62L þ and CTLA4 þ cells in draining lymph nodes of tumor-bearing mice injected with cells from the immunohigh pool (31) or

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immunolow pool (31) were not significantly different from each other (these data are not presented). Analogous findings were obtained if the tumor infiltrating cells were analyzed by RT-PCR for the expression of FoxP3, a unique transcription factor characteristic of T-reg cells.33,34 As indicated (Figure 3a), the intensity of the band for FoxP3 derived from tumors in mice injected with KLN205 cells followed by immunization with cells from the immunohigh pool (31) was less than that of the band for FoxP3 from tumors in mice injected with KLN205 cells followed by immunization with cells from the immunolow pool (31). The intensity of the bands derived from tumors in mice injected with KLN205 cells followed by cells from the non-enriched master pool or mice injected with KLN205 cells alone were not significantly different from each other. The same protocol was followed to compare the intensity of the band for FoxP3 derived from lymph nodes draining the site of injection of cells from the immunohigh pool (31) with the intensity of the band for FoxP3 derived from lymph nodes draining the site of injection of cells from the immunolow pool (31). As indicated (Figure 3b), the intensity of the band for FoxP3 was clearly less, if the cells were from the lymph nodes of draining the site of injection of cells from the immunohigh pool (31). As an additional control, the intensity of the band for FoxP3 in lymph nodes draining the site of injection of cells from the immunohigh pool (31) was compared with the intensity of the band for FoxP3 in lymph nodes draining the site of injection of cells from the

non-enriched master pool. In each instance, the least intense band was derived from lymph nodes draining the site of injection of cells from the immunohigh pool (31). Thus, T-reg cells were relatively deficient in squamous carcinomas undergoing regression and in the draining lymph nodes of mice immunized with the cells from the enriched, but not the non-enriched vaccines.

CD8 þ T cells were predominant in squamous carcinomas mice immunized with cells from the immunohigh pool (31) CD3/CD8 þ T cells are the major cell type activated for antitumor immunity in mice immunized with tumor vaccines.35,36 One possible explanation for the relative deficiency of T-reg cells in tumors in mice immunized with cells from the immunohigh pool (31) in a relative increase in the proportion of CD3/CD8 þ T cells. To investigate this question, the percentage of CD3/CD8 þ cells infiltrating squamous carcinomas in mice immunized with cells from the immunohigh pool (31) was compared with the percentage of CD3/CD8 þ cells in squamous carcinomas in mice immunized with cells from the immunolow pool (31) or the non-enriched master pool. Tumors were established in the left flank of naı¨ ve DBA/2 mice following an s.c. injection of 1 106 KLN205 cells. When the tumors were between 0.5 and 0.8 cm in diameter (6 days), the mice received a single s.c. injection of 4 106 cells from the immunohigh pool (31) in the right flank. Eleven days later, a tumor cell suspension was prepared and the percentage of CD3/CD8 þ cells was determined. As controls, the same procedure was followed except

Figure 3 FoxP3 þ cells were relatively deficient in squamous carcinomas in mice immunized with cells from the immunohigh pool (31). (a) Tumor infiltrating cells. Densitometry of RT-PCR bands for FoxP3 in squamous carcinomas derived from KLN205 cells in mice immunized with cells from the immunohigh pool (31), the immunolow pool (31) or the non-enriched master pool. One group of mice was injected with KLN205 cells alone. Pooled cell suspensions from two mice were analyzed. (b) Draining lymph nodes. Densitometry of RT-PCR bands for FoxP3 in lymph nodes draining the site of injection of cells from the immunohigh pool (31), the immunolow pool (31) or the non-enriched master pool. Lymph nodes from one group of mice injected with KLN205 cells alone were included as well. The protocol described in (a) was followed in (b) except that the tumors were analyzed by RT-PCR for FoxP3, as described in the Materials and methods section. (Densitometry determinations were performed in a BioRad Gel Doc.)

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that cells from the immunolow pool (31) or the nonenriched master pool were substituted for cells from the immunohigh pool (31). One group of mice received an injection of KLN205 cells alone. The least tumor volume was in mice immunized with the cells from the immunohigh pool (31) (Figure 4a). As indicated (Figure 4b), the highest percentage of CD3/CD8 þ cells (27.2%) was in tumors in mice immunized with cells from the immunohigh pool (31). It was significantly (Po0.001) greater than tumors from mice immunized with cells from the immunolow pool (31) (8.3%) or the non-enriched master pool (4.3%). The percentage of CD3/CD8 þ cells

in tumors in mice immunized with cells from the non-enriched master pool was not significantly different than in mice injected with KLN205 cells alone (Figure 4b). The same protocol was followed to determine the percent CD3/CD4 cells in infiltrating tumors derived from KLN205 SCC in mice injected with cells from the immunohigh pool (31), the immunolow pool (31) or the nonenriched master pool. The least percentage of CD3/CD4 cells was in tumors from mice injected with cells from the immunohigh or the immunolow pools (these data are not presented).

Figure 4 CD3/CD8 cells were predominant in squamous carcinomas in mice immunized with cells from the immunohigh pool (31). KLN205 cells (1 106) were injected s.c. into the right flank of DBA/2 mice. Six days later, the mice received a single s.c. injection into the left flank of 4 106 cells from the immunohigh pool (31). As controls, the same protocol was followed except that one group of mice was injected with an equivalent number of KLN205 cells alone, with non-enriched LM-IL-2Kb/KLN cells or with cells from the immunolow pool (31). The experiment was terminated 11 days after the injection of the vaccine. Tumor volumes were determined with a dial caliper by the formula 0.5 l w2. There were two mice in each group. The experiment was repeated three times with equivalent results. The protocol outlined in the legend to Figure 2 was followed except that mAbs for CD8 þ cells were substituted for mAbs for the T-reg cells.

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Inflammatory cell infiltrates failed to form in tumor-bearing mice treated by immunization with the DNA-based vaccine Sakaguchi et al.27,28 reported that autoimmunity developed in mice depleted of T-reg cells. To determine if autoimmunity developed in T-reg-deficient mice following immunization with the DNA-based vaccine, naive DBA/2 mice received three s.c. injections at weekly intervals of viable LM-IL-2KbKLN cells. One week after the last injection, the mice were euthanized and H and E sections, prepared from 10 different organs and tissues of the immunized mice, were examined for the presence of infiltrating cells. (The organs and tissues examined included the following: kidney, heart, brain, liver, spleen, duodenum, thyroid, lymph nodes and muscle obtained from two different sites.) For comparison, H and E sections prepared from the same types of organs and tissues of naı¨ ve mice were examined. The results (not presented) failed to reveal the presence of inflammatory cells in any instance.

Discussion

The vast majority of cancers of the head and neck are squamous carcinomas that arise in the oral cavity. It is the sixth most common malignancy. Approximately 40 000 new cases are diagnosed yearly in the United States and 500 000 worldwide.37 Despite advances in treatment, including surgery, radiation and chemotherapy, the longterm survival of patients with metastatic disease has remained essentially constant for the last 50 years.38 Eighty percent of patients with early stage oral squamous carcinoma survive 5 years after diagnosis; about 17% of patients with late stage disease survive for an equivalent period.39 Like cancers arising in various other organs and tissues, treatment at an early stage of the disease increases the likelihood of success. Various clinical trials are in progress designed to test immune-based therapies for patients with various types of malignancies including squamous cell carcinoma of the head and neck (SCCHN).40–42 However, not all cancer vaccines are equally successful in inducing therapeutic antitumor immune responses. Activated T-reg cells in the tumor-bearing host, which inhibit tumor immunity, are a severe impediment. In this report, we describe the characteristics of the cell populations infiltrating tumors derived from KLN205 cells, a highly aggressive squamous carcinoma cell line, following immunization with a unique DNA-based vaccine, which had been enriched for immunotherapeutic cells.18,35,43,44 The investigation was prompted by findings in multiple laboratories that T-reg cells, generated in tumor-bearing mice and patients, can inhibit antitumor immune responses induced by cancer vaccines.45–51 T cells from cancer patients that express CD4 þ CD25 þ , a membrane-associated determinant characteristic of T-reg cells, suppress the proliferation of CD4 þ CD25 T cells. In mice, studies indicate that removing T-reg cells

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blocking an immunoregulatory pathway induced by the inhibitory cells unmasks natural tumor immunosurveillance and augments the response to cancer vaccines. Studies of the corresponding T-cell populations in human cancer patients support a similar role for T-reg cells in suppressing antitumor immunity. In prior reports, we described the enhanced immunotherapeutic properties of a DNA-based vaccine, which had been enriched for immunotherapeutic cells. A series of steps were taken to increase the number of immunotherapeutic cells in the vaccine, that is, cells that induced immunity to the tumor. The rationale was that, like other cellular antitumor vaccines, the vast majority of cells in the non-enriched vaccine specified normal cellular constituents, which were unrelated to the induction of the antitumor immune response. The results indicated that the enrichment strategy significantly enhanced the immunotherapeutic properties of the vaccine. After three rounds of positive immune selection, robust immunity to squamous carcinoma was generated in mice with immunized with cells from the enriched vaccine. Still, as determined by the analysis presented in Figure 1, relatively few cells in the enriched vaccine were responsible for induction of the antitumor response. The proportion of immunotherapeutic cells after three rounds of positive immune selection was approximately 1 in 50 000. It is likely that amplification of the response in the immunized recipient was responsible. One immunotherapeutic cell may have indirectly induced multiple CTL through uptake of the cellular vaccine by DC of the tumor-bearing host. One possible explanation for the enhanced immunotherapeutic properties of the enriched vaccine was an alteration in the CD3/CD8/T-reg cell ratio. The percentage of tumor infiltrating cells that expressed the T-reg cell markers CD4/CD25 þ , CD4/CD62L, CD4/CTLA-4e and FoxP3 in SCC was significantly less in SCC in mice immunized with the enriched vaccine than in various control groups. At the same time, the percentage of CD3/ CD8 cells in the regressing tumors increased over sixfold, from 4.3% in tumors derived from KLN205 cells in mice immunized with the non-enriched vaccine to 27.2% in tumors in mice immunized with the enriched vaccine (Figure 4b). An increase in the proportion of CD3/CD8 cells in the regressing tumor was consistent with the enhanced immunotherapeutic properties of the enriched vaccine. It would be premature, however, to conclude that immunization with immunohigh cells (31) directly affected the number of T-reg cells. The absolute number of T-reg cells in the regressing tumors was not determined. Surprisingly, inflammatory cell infiltrates in various organs and tissues of the immunized mice were not detected. The immunohigh cells express normal cell constituents in largest proportion, suggesting that an alteration of the ratio CD3/CD8 cells, rather than a decrease in the absolute number of T-reg cells was responsible for the enhanced immunogenic properties of vaccine. The data presented reveal that after three rounds of positive immune selection the enrichment strategy re-


sulted in the generation of a vaccine of enhanced effectiveness in the treatment of mice with SCC. Conceivably, additional rounds of positive selection could further enhance the therapeutic properties of the vaccine, with a corresponding increase in the number of immunotherapeutic cells. It is likely that the strategy outlined here can be applied to enhance the immunotherapeutic properties of cellular vaccines for treatment of patients with SCCHN, and perhaps various other types of tumors as well. Acknowledgements

This work was supported by NIDCR Grant number 1 RO1 DEO13970-O1A2 awarded to Dr Cohen. The use of animals in these studies as reviewed and approved by the Animal Care Committee of the University of Illinois (Approval number 04-067, expires 7/07).

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