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The Journal of Immunology, 2004, 172: 5159-5167.
Copyright © 2004 by The American Association of Immunologists

Paracrine Release of IL-12 Stimulates IFN-{gamma} Production and Dramatically Enhances the Antigen-Specific T Cell Response after Vaccination with a Novel Peptide-Based Cancer Vaccine1

Mohamed L. Salem, Andre N. Kadima, Yuehua Zhou, Christophe L. Nguyen, Mark P. Rubinstein, Marina Demcheva, John N. Vournakis, David J. Cole and William E. Gillanders2

Department of Surgery, Section of Surgical Oncology, Medical University of South Carolina, Charleston, SC 29425


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Interleukin-12 can act as a potent adjuvant for T cell vaccines, but its clinical use is limited by toxicity. Paracrine administration of IL-12 could significantly enhance the response to such vaccines without the toxicity associated with systemic administration. We have developed a novel vaccine delivery system (designated F2 gel matrix) composed of poly-N-acetyl glucosamine that has the dual properties of a sustained-release delivery system and a potent adjuvant. To test the efficacy of paracrine IL-12, we incorporated this cytokine into F2 gel matrix and monitored the response of OT-1 T cells in an adoptive transfer model. Recipient mice were vaccinated with F2 gel/SIINFEKL, F2 gel/SIINFEKL/IL-12 (paracrine IL-12), or F2 gel/SIINFEKL plus systemic IL-12 (systemic IL-12). Systemic levels of IL-12 were lower in paracrine IL-12-treated mice, suggesting that paracrine administration of IL-12 may be associated with less toxicity. However, paracrine administration of IL-12 was associated with an enhanced Ag-specific T cell proliferative and functional response. Furthermore, paracrine IL-12 promoted the generation of a stable, functional memory T cell population and was associated with protection from tumor challenge. To study the mechanisms underlying this enhanced response, wild-type and gene-deficient mice were used. The enhanced immune response was significantly reduced in IFN-{gamma}–/– and IL-12R{beta}2–/– recipient mice suggesting that the role of IL-12 is mediated, at least in part, by host cells. Collectively, the results support the potential of F2 gel matrix as a vaccine delivery system and suggest that sustained paracrine release of IL-12 has potential clinical application.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Interleukin-12 is a potent cytokine central to the regulation of innate and adaptive immune responses (1). It exerts a number of regulatory effects on T lymphocytes and NK cells (1, 2, 3). These include facilitating specific CD8+ T cell responses, promoting the development of Th1-type T cells (4, 5, 6, 7, 8, 9, 10), enhancing the lytic activity of NK cells, and inducing the secretion of IFN-{gamma} by both T cells and NK cells (2). The activation of T cells by IL-12, in addition to the production of IFN-{gamma}, appears to underlie the well-known antitumor effects of IL-12. In fact, systemic administration of IL-12 has been shown to significantly suppress the growth of a variety of established murine tumors, prolonging the survival of tumor-bearing mice (11, 12, 13).

Despite the efficacy of IL-12, the best approaches for delivery of IL-12 in vivo remain to be determined. Although the pharmacokinetics of IL-12 are more favorable than those of many other cytokines, repeated systemic administration is required for maximal therapeutic activity in mice (14, 15). The systemic administration of IL-12 is known to be associated with significant toxicity, and this toxicity has essentially precluded its use in clinical practice (16, 17). Alternative approaches for IL-12 delivery include viral-mediated gene therapy and gene-modified tumor cell vaccines. Unfortunately, viral-mediated gene therapy is associated with the generation of neutralizing Abs (18, 19), and gene-modified tumor cell vaccines lack the simplicity and the versatility required for universal clinical application (18).

The development of a reliable and inexpensive technology for the sustained local delivery of cytokines and other biologic response modifiers at the site of a peptide-based cancer vaccine could facilitate the clinical integration of IL-12. We have isolated a novel polysaccharide polymer (poly-N-acetyl glucosamine) that can be formulated into a biocompatible gel (F2 gel). F2 gel has the capacity to provide sustained local delivery of antigenic peptides and cytokines, as well as being a potent immune adjuvant (20, 21). Formation of an emulsion suitable for vaccination does not require vigorous sonication, and labile proteins are efficiently incorporated without denaturation. We have previously shown that incorporation of GM-CSF into this modular vaccine enhances the Ag-specific T cell response (21). In the present study, we investigated whether paracrine administration of IL-12 is a viable delivery strategy, potentially limiting the toxicity associated with systemic administration of this cytokine. To precisely define the response of Ag-specific T cells to this novel vaccine, we used a murine adoptive transfer model system based on the OT-1 TCR transgenic mouse (21).

To our knowledge, this is the first study to compare the effect of paracrine and systemic IL-12 on Ag-specific T cell responses. Paracrine IL-12 was clearly superior to systemic IL-12, enhancing T cell proliferation and function, and promoting the formation of a significant memory population. Furthermore, using gene-deficient recipient mice, we examined the mechanisms of action of paracrine IL-12. Using IFN-{gamma}-deficient and IL-12R{beta}2-deficient mice, we show that the effect of paracrine IL-12 is mediated in part by an increase in host IFN-{gamma} production by APCs and other cells of the innate immune system. Most important, however, the data clearly show that a single dose of IL-12 loaded into F2 gel and delivered at the site of vaccination is equivalent to the expected activity of multiple doses of IL-12 administered systemically. Paracrine administration of IL-12 at the site of vaccination is thus a simple form of delivery that is safe, efficient, economical, and promising for application in the clinical setting.


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

OT-1 TCR transgenic mice (V{alpha}2/V{beta}5) were purchased from The Jackson Laboratory (Bar Harbor, ME) and were bred with B6.SJL mice to generate Ly5.1/Ly5.1 mice heterozygous for the OT-1 TCR transgene. Transgene status was confirmed by flow cytometry with mAb specific for V{alpha}2. Congenic C57BL/6 mice were used as recipients, and in certain experiments, IFN-{gamma}-deficient (B6.129S7tm/tm) or IL-12R{beta}2-deficient (B6.129S7tm/jm) mice were used as recipients. All animals were housed under specific pathogen-free conditions in accordance with National Institutes of Health guidelines (Bethesda, MD).

Cell lines

EL-4 is a thymoma derived from the C57BL/6 mouse (H-2b). The OVA-transfected B16 melanoma tumor cell line (B16-OVA) was kindly provided by Dr. R. Dutton (Trudeau Institute, Saranac Lake, NY). Cells were maintained in vitro in complete RPMI medium (RPMI 1640, 0.1% penicillin/streptomycin (Cellgro, Herndon, VA), 10% FBS (HyClone Laboratories, Logan, UT), 0.2% L-glutamine (Sigma-Aldrich, St. Louis, MO), 0.05% 2-mercaptoethanol, 0.01% sodium pyruvate (Life Technologies, Grand Island, NY), 0.1% HEPES, and 0.1% nonessential amino acids).

Adoptive transfer of OT-1 T cells

Spleen and lymph nodes (cervical, axillary, brachial, inguinal, and mesenteric) from OT-1 TCR transgenic mice were harvested, homogenized, and washed in HBSS (Cellgro). Pooled cells were then passed over a CD8-negative selection column from R&D Systems (Minneapolis, MN). CD8+/Ly5.1+ OT-1 T cells were i.v. transferred into naive C57BL/6 Ly5.2 recipient mice (1.5 x 106 cells/mouse).

Vaccination

Recombinant murine IL-12 and GM-CSF were purchased from R&D Systems and reconstituted in 0.1% BSA (Sigma-Aldrich). SIINFEKL peptide was purchased from American Peptide (Sunnyvale, CA) and reconstituted in 10% DMSO (Sigma-Aldrich). A specific poly-N-acetyl glucosamine (p-GlcNAc) gel formulation, referred to as F2 gel matrix, was designed to optimize immune system activation. The F2 gel matrix was prepared by chemical deacetylation of p-GlcNAc to 70% with conversion of the polymer to a lactate salt. A final 5% polymer concentration was achieved by hydration and mixed with a 10% DMSO/SIINFEKL/cytokine solution to form the F2 gel/SIINFEKL/cytokine matrix. Mice were vaccinated s.c. at the base of the tail with 150 µl of F2 gel/SIINFEKL/cytokine matrix containing 100 µg of SIINFEKL and 1.5 µg of IL-12 or GM-CSF. In some experiments, mice were vaccinated s.c. with F2 gel/SIINFEKL matrix alone or with systemic administration of a single dose of IL-12 (1.5 µg). For secondary challenge, mice were challenged with F2 gel/SIINFEKL matrix at the base of the tail.

Monoclonal Abs and flow cytometry

Anti-CD16/CD32, anti-Ly5.1-FITC, anti-CD8-CyChrome, and anti-CD62L-PE were purchased from BD Pharmingen (San Diego, CA). The PBL, draining lymph node, and spleen of vaccinated mice were analyzed at various time points after vaccination. Single-cell suspensions were prepared and 1 x 106 cells were treated with anti-CD16/CD32 for 5 min on ice. Cells were then stained with the indicated mAb. After a 30-min incubation on ice, the cells were washed twice, and resuspended in 0.3 ml of 0.5% BSA, 0.02% sodium azide solution. Cells were analyzed by flow cytometry using the CellQuest software package (BD Biosciences, San Jose, CA).

Measurement of IL-12p70 in serum

B6 mice, six per group, were adoptively transferred with 1.5 x 106 enriched CD8+ OT-1 cells. After 24 h, mice were vaccinated at the base of the tail (s.c.) with F2 gel/SIINFEKL matrix alone, F2 gel/SIINFEKL/IL-12 (paracrine IL-12), or F2 gel/SIINFEKL with systemic administration of IL-12. Vaccinated mice were bled at multiple time points and sera were isolated for measurement of IL-12p70 by ELISA (R&D Systems), and IFN-{gamma} by cytometric bead array (BD Pharmingen).

Cytotoxicity assay

The lytic activities of PBL, draining lymph node cells, and spleen cells obtained on day 7 from control or experimental mice were determined using a standard 4-h 51Cr release assay. Serial dilutions of effector cells were plated in 96-well U-bottom plates with 5 x 103 51Cr-labeled EL-4 cells, or labeled EL-4 cells pulsed with SIINFEKL peptide. After 4-h incubation at 37°C, 25 µl of culture supernatant was removed from each well and radioactivity was determined. The percentage of specific 51Cr release was calculated according to the following equation: percentage of specific lysis = [(experimental release – spontaneous release)/(maximum release – spontaneous release)] x 100. Maximum target release was determined by treatment of cells with 9% Triton X-100 solution (Sigma-Aldrich).

Cell proliferation and cytokine production

The PBL, draining lymph node, and spleen of control and experimental mice were harvested on day 7. Single-cell suspensions were prepared in complete RPMI 1640 medium and 10 x 106 (spleen and draining lymph node) or 1 x 106 (PBL) effector cells were cocultured with fresh, irradiated (2000 rad) splenocytes (20 x 106) from naive syngeneic mice to which SIINFEKL peptide (5 µg/ml) was added. For memory cells, draining lymph node cells were harvested on days 3 and 7 post secondary challenge, and 1 x 105 cells were cultured with fresh irradiated (2000 rad) splenocytes (1 x 105) from naive syngeneic mice pulsed with or without 5 µg/ml SIINFEKL peptide. Supernatants were collected 24 h later and levels of IFN-{gamma} and TNF-{alpha} were measured by cytometric bead array (BD Pharmingen). To assess activity of OT-1 cells for Ag recall, vaccinated mice were rechallenged with F2 gel/SIINFEKL on day 40 and sacrificed 3 days later. Draining lymph node cells were harvested, and 1 x 105 cells were cultured with fresh irradiated (2000 rad) splenocytes (1 x 105) from naive syngeneic mice pulsed with or without 5 µg/ml SIINFEKL peptide. After 24 h cell proliferation was measured by MTT assay as previously described (22).

Ag presentation assay

For preparation of APCs, mice were transferred and vaccinated as previously described, and spleen cells were harvested from the experimental groups on day 3 after priming. Cells were irradiated at 3 Gy, and pulsed for 1 h at 37°C with different concentrations of SIINFEKL. Responder Ag-specific CD8+ T cells were enriched with a CD8 column (R&D Systems) using spleens and lymph nodes of naive OT-1 mice. Responder CD8+ OT-1 cells were cocultured in triplicate with or without SIINFEKL-pulsed APCs at 37°C. After 24 h, 50 µl of the culture supernatants were collected and stored at –20°C for cytokine analysis. Then, the culture assay was pulsed with [3H]thymidine (1 µCi = 37 kBq) and cells were harvested 6 to 8 h later. Thymidine incorporation into DNA was measured as counts per minute on a Packard Matrix 96 beta counter. The levels of IL-2, IFN-{gamma}, and TNF-{alpha} in the supernatants were measured by cytometric bead array (BD Pharmingen).

Tumor challenge

Naive C57BL6 mice (n = 7) were challenged with 1 x 106 B16-OVA cells by tail-vein injection. Two weeks following tumor challenge, mice were adoptively transferred with 1.5 x 106 enriched CD8+ OT-1 cells. After 24 h adoptive transfer, mice were vaccinated at the base of the tail with F2 gel/SIINFEKL, F2 gel/SIINFEKL plus systemic IL-12, F2 gel/SIINFEKL/IL-12, or a sham vaccine. Mice were sacrificed 28 days after tumor challenge and the number of pulmonary metastases was determined in a blinded fashion after fixing the lung in 1% paraformaldehyde (Sigma-Aldrich).


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Incorporation of IL-12 into F2 gel alters the pharmacokinetics of IL-12 administration

To assess the efficacy of F2 gel as a delivery vehicle for IL-12 in vivo, we compared the pharmacokinetics of IL-12 following vaccination with F2 gel/SIINFEKL/IL-12 or F2 gel/SIINFEKL with systemic administration of IL-12. After vaccination, serum samples were obtained at serial time points, and IL-12 p70 levels were measured by ELISA. After systemic administration, IL-12 levels peaked at 15 min, and dropped rapidly to minimal but measurable levels by 4 h (Fig. 1). In contrast, when IL-12 was incorporated into F2 gel, serum levels of IL-12 were low but measurable at all time points. These data confirm that incorporation of IL-12 alters the pharmacokinetics of IL-12, and suggest that this route of administration may avoid the toxicity associated with elevated systemic levels of IL-12. As detailed below, incorporation of IL-12 dramatically enhances the efficacy of this vaccine, suggesting that the route of administration of this cytokine may be as important as dose.



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  		FIGURE 1. Incorporation of IL-12 into the F2 gel vaccine profoundly alters the pharmacokinetics of IL-12. A total of 1.5 x 106 OT-1 T cells were adoptively transferred into C57BL/6 mice (n = 6 mice per group). Recipient mice were then vaccinated with F2 gel/SIINFEKL/IL-12 or F2 gel/SIINFEKL with concomitant systemic administration of IL-12. Mice were then bled at the indicated time points following vaccination, and serum levels of IL-12 were determined by ELISA. An inset with exploded y-axis scale to show the levels of IL-12. The results are representative of two independent experiments.

 
Incorporation of IL-12 into F2 gel vaccine significantly enhances the Ag-specific T cell proliferative response

To precisely define the CD8+ T cell response to F2 gel vaccine, we used an adoptive transfer model based on the OT-1 TCR transgenic mouse. OT-1 T cells recognize the SIINFEKL peptide in the context of the MHC class I molecule H-2Kb (23). Naive CD8+ OT-1 T cells (1.5 x 106) were adoptively transferred into age- and gender-matched congenic recipients, and this population was precisely monitored by flow cytometry with anti-Ly5.1 and anti-CD8 mAb. After adoptive transfer, this Ag-specific population represented ~0.2–0.4% of cells in the lymphoid organs. Mice were rested for 1 day after adoptive transfer and then vaccinated at the base of the tail with the specified F2 gel vaccines.

We have previously shown that incorporation of GM-CSF into F2 gel vaccine enhances the primary Ag-specific T cell response (21). To assess whether other cytokines might provide a similar beneficial effect, we incorporated IL-12, TNF-{alpha}, IL-18, macrophage-inflammatory protein 1{alpha}, and secondary lymphoid chemokine into the F2 gel vaccine. Flow cytometry was used to quantitate the percentage of OT-1 T cells in the peripheral blood at serial time points after vaccination. Incorporation of IL-12, but not other cytokines, significantly enhanced the primary Ag-specific, T cell response (Fig. 2A and data not shown). Specifically, incorporation of IL-12 increased the percentage of OT-1 T cells in the PBL at all time points from days 3–14. This increased percentage of OT-1 T cells persisted until 150 days (data not shown). To control for possible systemic release of IL-12, this experiment was repeated with IL-12 incorporated into the F2 gel (F2 gel/SIINFEKL/IL-12), and compared with systemic administration of an equivalent dose of IL-12 (F2 gel/SIINFEKL with systemic IL-12). Again, incorporation of IL-12 into F2 gel vaccine resulted in dramatic increase in the number of Ag-specific T cells during the primary response. However, vaccination with concomitant administration of systemic IL-12 did not enhance the response (Fig. 2B).



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  		FIGURE 2. Incorporation of IL-12 into the F2 gel vaccine significantly enhances the primary Ag-specific T cell response. A total of 1.5 x 106 OT-1 T cells were adoptively transferred into C57BL/6 mice (n = 4 mice per group). Recipient mice were then bled at the indicated time points following vaccination and the percentage of OT-1 T cells in PBL samples was determined by two-color flow cytometry (anti-Ly5.1-FITC and anti-CD8-CyChrome). A, Results from mice vaccinated with a sham vaccine, F2 gel/SIINFEKL/IL-12, and F2 gel/SIINFEKL/GM-CSF. B, Results from mice vaccinated with a sham vaccine, F2 gel/SIINFEKL/IL-12, or F2 gel/SIINFEKL with concomitant systemic administration of IL-12. The results are representative of three independent experiments.

 
To further define the primary response to F2 gel vaccine, we determined the percentage of OT-1 T cells in different lymphoid compartments at serial time points after vaccination. Days 3 and 7 were chosen for these analyses based on data that suggest that the Ag-specific T cell response peaks in the draining lymph node and PBL at these time points, respectively (data not shown). Incorporation of IL-12 into F2 gel vaccine markedly increased the percentage of OT-1 T cells in the draining lymph node and PBL on day 3, and in the PBL on day 7 (Fig. 3). At day 3, the majority of Ag-specific T cells were in the draining lymph node (Fig. 3A). At day 7, the percentage of OT-1 T cells was decreased in the draining lymph node in all experimental groups, and the majority of Ag-specific T cells were present in the PBL (Fig. 3B). This stepwise progression from the draining lymph node to the PBL suggests that the F2 gel serves as an effective depot, preventing Ag and cytokine from being immediately released systemically.



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  		FIGURE 3. Enhanced primary Ag-specific T cell response is evident in all lymphoid compartments. A total of 1.5 x 106 OT-1 T cells were adoptively transferred into C57BL/6 mice (n = 4 mice per group). Recipient mice were then vaccinated with F2 gel/SIINFEKL, F2 gel/SIINFEKL/IL-12, or F2 gel/SIINFEKL with concomitant systemic administration of IL-12. Mice were sacrificed on day 3 (A) and day 7 (B) following vaccination and the lymphoid organs were harvested (PBL, spleen, and draining lymph node). The percentage of OT-1 T cells was determined by two-color flow cytometry (anti-Ly5.1-FITC and anti-CD8-CyChrome). The results are representative of two independent experiments.

 
Incorporation of IL-12 into the F2 gel vaccine significantly enhances Ag-specific T cell function

To define the functional capacity of Ag-specific T cells after vaccination, cytolytic activity and cytokine production were measured ex vivo. OT-1 T cells were adoptively transferred into congenic recipients, and these animals were then vaccinated with F2 gel/SIINFEKL, F2 gel/SIINFEKL/IL-12, F2 gel/SIINFEKL with concomitant systemic administration of IL-12, or a sham PBS vaccine. The lymphoid organs of experimental animals were harvested on day 7 after vaccination, and a single-cell suspension was prepared. Cytolytic activity was then measured directly ex vivo (no in vitro stimulation before the assay) using SIINFEKL-pulsed EL4 cells in a standard 4-h 51Cr release assay (Fig. 4). Incorporation of IL-12 into the F2 gel vaccine substantially increased the lytic activity of Ag-specific T cells in the PBL (Fig. 4A), spleen (Fig. 4B), draining lymph node, and liver (data not shown). No nonspecific lysis of unpulsed EL4 cells was detectable (data not shown). Vaccination with F2 gel/SIINFEKL, or F2 gel/SIINFEKL with concomitant systemic administration of IL-12, resulted in only marginal increases in lytic activity, confirming that at equivalent doses paracrine administration of IL-12 is superior to systemic administration of IL-12 in terms of inducing T cell function.



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  		FIGURE 4. Paracrine administration of IL-12 enhances the lytic capacity of Ag-specific T cells. A total of 1.5 x 106 OT-1 T cells were adoptively transferred into C57BL/6 mice (n = 4 mice per group). Recipient mice were then vaccinated with a sham vaccine, F2 gel/SIINFEKL, F2 gel/SIINFEKL/IL-12, or F2 gel/SIINFEKL with concomitant systemic administration of IL-12. The PBL (A) and spleen (B) were harvested on day 7 and lytic capacity was determined by a standard 51Cr release assay using SIINFEKL-pulsed EL-4 targets. No significant lysis of control EL-4 targets was observed in any condition. The results are representative of two independent experiments.

 
IFN-{gamma} and TNF-{alpha} production are associated with cytotoxic T lymphocyte function (24), and IFN-{gamma} production in particular is known to be associated with antitumor immunity. Production of these cytokines is therefore considered to be an index of T cell function. To determine the ability of F2 gel vaccine to induce the capacity for cytokine production in responding T cells, adoptive transfer experiments were performed. Recipient mice were vaccinated as previously described. PBL, spleen, and draining lymph node were harvested on day 7, and single-cell suspensions were cultured with SIINFEKL-pulsed, irradiated EL-4 cells for 24 h. Supernatants were collected and levels of IFN-{gamma} and TNF-{alpha} were measured by cytometric bead array. Consistent with previous results, effector cells from mice vaccinated with F2 gel/SIINFEKL/IL-12 produced higher levels of IFN-{gamma} and TNF-{alpha} (Table I). However, effector cells from mice vaccinated with F2 gel/SIINFEKL/IL-12 produced substantially higher levels of IFN-{gamma} (Table I), suggesting that the functional differences observed are not simply related to enhanced proliferation or cell number.


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  		Table I. Paracrine IL-12 enhances production of both IFN-{gamma} and TNF-{alpha}a

 
Incorporation of IL-12 into F2 gel vaccine enhances the establishment of an Ag-specific memory T cell population

The results discussed indicate that incorporation of IL-12 into the F2 gel vaccine significantly enhances the primary Ag-specific T cell response. To assess the effect of vaccination on T cell memory, flow cytometry and functional analyses were performed before and after F2 gel/SIINFEKL rechallenge at day 40. This time point is considered to be an indicator of the memory CD8 T cell response (25). Before rechallenge, the percentage of OT-1 T cells in the PBL was determined by flow cytometry. Paracrine administration of IL-12 substantially increased the Ag-specific memory T cell population in PBL (Fig. 5A), as evidenced by the persistent down-regulation of CD62 ligand expression (Fig. 5B) (26). Mice from all experimental groups were then challenged with F2 gel/SIINFEKL, and draining lymph node cells were harvested on days 3 and 7 to determine capacity for proliferation and cytokine production. Single-cell suspensions were cultured with SIINFEKL-pulsed, irradiated syngeneic spleen cells for 24 h. Then both proliferation and levels of IFN-{gamma} were measured. Effector cells from mice initially vaccinated with F2 gel/SIINFEK/IL-12 showed more proliferation (Fig. 5C) and IFN-{gamma} production when revaccinated with F2 gel/SIINFEKL (Table II). These results show that treatment with paracrine IL-12 establishes long-lived memory CD8 T cells.



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  		FIGURE 5. Incorporation of IL-12 into the F2 gel vaccine enhances the establishment of a memory T cell population. A total of 1.5 x 106 OT-1 T cells were adoptively transferred into C57BL/6 mice (n = 4 mice per group). Recipient mice were then vaccinated with a sham vaccine, F2 gel/SIINFEKL, F2 gel/SIINFEKL/IL-12, or F2 gel/SIINFEKL with concomitant systemic administration of IL-12. At day 40, mice were bled and the number (A) and phenotype (B) of Ag-specific T cells were determined by flow cytometry. C, Vaccinated mice were rechallenged with F2 gel/SIINFEKL on day 40 after primary vaccination, and then mice were killed 3 days later. Draining lymph nodes were harvested and draining lymph node cells (1 x 105) were cocultured with different numbers of syngeneic irradiated splenocytes pulsed with 5 µg/ml SIINFEKL peptide. After 24 h proliferation of cultured cells was measured by MTT assay. The results are representative of two independent experiments.

 

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  		Table II. Paracrine IL-12 enhances IFN-{gamma} production by memory T cells

 
Incorporation of IL-12 into F2 gel vaccine is associated with protection from tumor challenge

To determine whether the enhanced Ag-specific T cell response following F2 gel/SIINFEKL/IL-12 vaccine is associated with antitumor immunity, tumor challenge experiments were performed. Naive mice were challenged with 1 x 106 B16-OVA melanoma by tail vein injection and then transferred with OT-1 cells 2 wk later. Sentinel mice were killed before adoptive transfer to confirm establishment of colonies of B16-OVA in the lung. Then, mice were vaccinated with sham vaccine (PBS), F2 gel/SIINFEKL, F2 gel/SIINFEKL/IL-12, or F2 gel/SIINFEKL plus systemic IL-12. Two weeks after vaccination (4 wk after tumor challenge), the mice were sacrificed and lungs were harvested. The specimens were then coded so that the number of lung metastases could be determined in a blinded fashion. Lung metastases were determined by gross evaluation using microscope slides to enhance visualization of the lung parenchyma. We found that only immunization with F2 gel/SIINFEKL/IL-12 markedly reduced the number of tumor colonies in the lung (Table III). We conclude from these results that the simultaneous release of IL-12 and peptide from F2 gel is necessary to optimize the therapeutic effect against established tumor.


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  		Table III. Paracrine IL-12 supresses tumor growth

 
Paracrine administration of IL-12 enhances activation and function of APCs

To explore the mechanisms of action of IL-12, we designed experiments aimed at determining whether incorporation of IL-12 into the F2 gel vaccine enhances activation of APCs. Although there is evidence to suggest that systemic administration of IL-12 can act directly on T cells, our hypothesis is that paracrine administration of this cytokine serves to preferentially target APCs (see Discussion). To assess the function of APCs after vaccination we developed an ex vivo assay system. Recipient mice were vaccinated with F2 gel/SIINFEKL, F2 gel/SIINFEKL/IL-12, F2 gel/SIINFEKL with concomitant systemic administration of IL-12, or a sham PBS vaccine. Four days after vaccination, mice were sacrificed, spleens were harvested and a single-cell suspension was prepared. Splenocyte preparations were pulsed with varying concentrations of SIINFEKL peptide and then cocultured with naive OT-1 T cells. Splenocytes obtained from mice vaccinated with F2 gel/SIINFEKL/IL-12 were more effective in stimulating naive OT-1 T cells as measured by thymidine incorporation (Fig. 6A) and cytokine production (Fig. 6B).



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  		FIGURE 6. Paracrine administration of IL-12 enhances activation and function of APCs. A total of 1.5 x 106 OT-1 T cells were adoptively transferred into C57BL/6 mice (n = 4 mice per group). Recipient mice were then vaccinated with a sham vaccine, F2 gel/SIINFEKL, F2 gel/SIINFEKL/IL-12, or F2 gel/SIINFEKL with concomitant systemic administration of IL-12. A, Splenocytes were harvested from experimental mice 3 days following vaccination. Irradiated splenocytes were pulsed with 0.05–5 µg SIINFEKL and then cocultured with 5 x 104 naive OT-1 T cells. Proliferation of OT-1 T cells was measured by [3H] uptake. B, Supernatants were collected from the cultures indicated and IFN-{gamma}, IL-2, and TNF-{alpha} production was determined by flow cytometric bead array. The results are representative of two independent experiments.

 
Enhanced immune response associated with paracrine administration of IL-12 is dependent on IL-12R expression on recipient APCs

IL-12 is a potent cytokine and is known to interact with both T cells and APCs. To directly assess the role of APCs in enhancing the immune response after paracrine administration of IL-12 in vivo, we performed parallel adoptive transfer experiments into wild-type and IL-12R{beta}2–/– mice. Naive OT-1 T cells were adoptively transferred into wild-type and IL-12R{beta}2–/– mice. Recipient mice were then vaccinated with F2 gel/SIINFEKL, F2 gel/SIINFEKL/IL-12, F2 gel/SIINFEKL with concomitant systemic administration of IL-12, or a sham PBS vaccine. The mice were then bled at serial time points and the percentage of Ag-specific T cells was determined by flow cytometry. Consistent with previous experiments, paracrine administration of IL-12 significantly enhanced the primary immune response in wild-type recipients (Fig. 7A). However, this enhanced response was significantly reduced in IL-12R{beta}2–/– recipient mice, particularly at later time points. To define the impact on memory cells, mice were sacrificed at day 60 and the percentage of Ag-specific T cells was determined in different lymphoid organs (Fig. 7B). In IL-12R{beta}2–/– recipient mice, almost no memory cell population is present. Collectively, these data indicate that the dramatic benefit of paracrine administration of IL-12 is largely dependent on activation of recipient APCs.



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  		FIGURE 7. The enhanced immune response associated with incorporation of IL-12 into the F2 gel vaccine is at least partially dependent on IL-12 interaction with recipient APCs. A total of 1.5 x 106 OT-1 T cells were adoptively transferred into wild-type or IL-12R{beta}2–/– mice (n = 4 mice per group). Mice were then vaccinated with F2 gel/SIINFEKL/IL-12. A, Mice were bled at the indicated time points following vaccination and the percentage of OT-1 T cells was determined by flow cytometry. B, Mice were sacrificed at day 60 following vaccination and the lymphoid organs were harvested. The percentage of OT-1 T cells was determined by flow cytometry.

 
Host IFN-{gamma} production is a mediator of the enhanced immune response associated with paracrine administration of IL-12

Previous studies have shown that many, but not all, of the in vivo effects of IL-12 can be attributed to the induction of IFN-{gamma} production (27). To study the role of IFN-{gamma} in mediating the enhanced response to paracrine administration of IL-12, parallel adoptive transfer experiments were performed into wild-type and IFN-{gamma}–/–-deficient mice. Recipient mice were vaccinated with F2 gel/SIINFEKL, F2 gel/SIINFEKL/IL-12, F2 gel/SIINFEKL with concomitant systemic administration of IL-12, or a sham PBS vaccine. The percentage of OT-1 T cells was then determined at multiple time points in PBL. Consistent with the experiments previously detailed, incorporation of IL-12 into the F2 gel vaccine dramatically enhanced the primary immune response. However, the response to the F2 gel/SIINFEKL/IL-12 vaccine was clearly attenuated in IFN-{gamma}–/–-deficient mice (Fig. 8), suggesting that the beneficial effect of paracrine administration of IL-12 is dependent on IFN-{gamma} production by the host.



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  		FIGURE 8. The enhanced immune response following paracrine administration of IL-12 is dependent on host IFN-{gamma} production. A total of 1.5 x 106 OT-1 T cells were adoptively transferred into wild-type or IFN-{gamma}–/– mice (n = 4 mice per group). Recipient mice were then vaccinated with F2 gel/SIINFEKL, F2 gel/SIINFEKL/IL-12, or F2 gel/SIINFEKL with concomitant systemic administration of IL-12. A, Wild-type and IFN-{gamma}–/– mice were bled at the indicated time points following vaccination and the percentage of OT-1 T cells was determined by flow cytometry. (For clarity, only the results of mice vaccinated with F2 gel/SIINFEKL/IL-12 are shown; the other conditions showed no significant response, similar to results presented in Fig. 2). B, Peripheral blood was obtained at day 60 following vaccination. The percentage of OT-1 T cells was determined by flow cytometry.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
Although systemic administration of IL-12 is known to be a potent T cell adjuvant (4, 5, 9, 28, 29, 30) it is also associated with dose- and schedule-dependent toxicity in humans (10, 31, 32, 33, 34). In fact, the toxicity of IL-12 has been the major limitation in translating this potent cytokine to the clinical arena. Recent research has focused on altering the schedule and dose of IL-12 to decrease its toxicity, but such regimens are associated with a concomitant decrease in efficacy (31, 35, 36). Tumor cell vaccines genetically engineered to produce IL-12 have yielded antitumor immunity in mice (18, 37, 38). Such an approach, however, may not be clinically feasible because it is labor-intensive, and autologous tumor is not always readily available (18, 39). To define a more universal alternative for cytokine immunotherapy, we explored the potential of a novel polysaccharide polymer, F2 gel, to serve as a modular vaccine adjuvant and cytokine delivery vehicle.

In the present work, we have shown that F2 gel can effectively deliver biologically active IL-12 at the vaccine site. This paracrine administration of IL-12 dramatically enhanced the Ag-specific T cell response compared with vaccination alone, or vaccination in combination with systemic administration of IL-12. Specifically, evaluation of the proliferative response by flow cytometry at serial time points after vaccination documented that the peak was significantly increased, and that the duration of the response was considerably prolonged with paracrine administration of IL-12 (Figs. 1 and 3). Furthermore, analyses of T cell function using ex vivo 51Cr release assays (Fig. 4) and assays for IFN-{gamma} production (Table I) confirmed the efficacy of this vaccine strategy (lytic capacity, and in particular, the ability to produce IFN-{gamma} are known to be associated with antitumor immunity). The clinical relevance of these findings was confirmed by the observation that paracrine administration of IL-12 significantly enhanced antitumor immunity in a murine melanoma lung metastasis model (Table III).

The current literature suggests that IL-12 may act directly on CD8+ T cells, or may act indirectly by stimulating APCs and/or NK cells. Evidence that IL-12 can directly stimulate CD8+ T cells includes in vitro studies using microspheres as artificial APCs (9), and in vivo studies using IL-12R{beta}1 gene-deficient mice (4). Evidence that IL-12 can directly stimulate APCs includes studies of CD8{alpha}– splenic dendritic cells. In these studies, IL-12 stimulation results in NF-{kappa}B-mediated signaling, increased maturation of dendritic cells, and enhanced APC function (40, 41). Because of these divergent potential mechanisms of action, we were interested in studying the mechanisms of IL-12 in our model system, after paracrine administration of IL-12. We provide evidence that paracrine administration of IL-12 increased the number of dendritic cells in the spleen and PBL, and enhanced the ability of APCs in the spleen to present SIINFEKL peptide to naive OT-1 cells ex vivo (Fig. 6). To further define the contribution of APCs, we performed experiments after adoptive transfer into wild-type and IL-12R{beta}2–/– recipient mice. We found that when adoptive transfer was performed into IL-12R{beta}2 gene-deficient mice, the peak of the primary response was significantly reduced (Fig. 7). Although these results indicate that the effects of paracrine administration of IL-12 are mediated at least in part by direct stimulation of host APCs, one cannot exclude other possibilities. For instance, it has been reported that functions of NK and NK T cells are induced by IL-12 (1, 42), and that these cells are major contributors for the adjuvant effects of IL-12 (43, 44, 45, 46, 47, 48). Because NK cells express IL-12R{beta} (49), and IL-12R{beta}2–/– mice are deficient in their ability to produce IFN-{gamma} (50), it is possible that the observed reduction of CD8 T cell response in IL-12R{beta}2–/– mice could also be due to the lack of endogenous IFN-{gamma} produced by NK cells in response to IL-12. In line with this suggestion, we have found a significant increase in the number of NK cells in PBL and liver in F2 gel/SIINFEKL/IL-12 immunized mice (data not shown). Thus, IL-12 may act primarily on NK and/or NK T cells, which in turn activate dendritic cells. Consistent with this hypothesis, it has been demonstrated a critical interaction between Ly49H+ NK cells and CD8{alpha}+ dendritic cells (51), whereby the presence of Ly49H+ NK cells results in maintenance of CD8{alpha}+ dendritic cells in the spleen during acute murine CMV infection (52). It is also possible that other host cells such as macrophages and neutrophils may contribute to the adjuvant effects of IL-12 seen in our model. This is possible as these two populations have been reported to mediate the antitumor effects of IL-12 (53, 54, 55, 56, 57, 58). Further studies are required to define the reciprocal interaction of these cells in the adjuvant effects of IL-12.

The effects of IL-12 have been found to be IFN-{gamma}-dependent in several model systems (27, 59, 60). In our studies, the efficacy of IL-12 is significantly reduced after adoptive transfer into IL-12R{beta}2–/– mice; these mice are known to be deficient in their ability to produce IFN-{gamma} (61). Taken together, these data suggest that IFN-{gamma} is a critical mediator in the response to IL-12. To directly address this hypothesis, we investigated the role of IFN-{gamma} by defining the CD8+ T cell response in wild-type and IFN-{gamma} gene-deficient mice. We found that the Ag-specific T cell response was significantly reduced in IFN-{gamma} gene-deficient mice (Fig. 8). Although the initial response appeared to be similar, the peak and memory responses were significantly attenuated in IFN-{gamma} gene-deficient mice, implicating an important role for IFN-{gamma} produced by host cells. Although the role of IFN-{gamma} in the generation of memory CD8+ T cells has not been extensively studied, evidence in the literature suggests that IFN-{gamma} plays an important role in this process. For instance, the ability of systemic IL-12 to induce bystander proliferation of adoptively transferred CD44high CD8+ T cells in the absence of Ag was highly dependent on host IFN-{gamma} (62). Further, induction of bystander CD8+ T cell proliferation by IL-18 is largely IFN-{gamma}-dependent, consistent with the hypothesis that cytokines capable of inducing IFN-{gamma} promote the turnover of memory phenotype CD8+ T cells (63). Overall, these results support our hypothesis that IFN-{gamma} produced by host APCs and NK cells plays a crucial role after paracrine administration of IL-12, and is consistent with the hypothesis that IFN-{gamma} is the final effector cytokine for induction of Ag-specific T cell memory.

In this report we have established that paracrine administration of IL-12 as a component of the F2 gel vaccine is superior to systemic administration of IL-12 as measured by multiple immunologic parameters including Ag-specific T cell proliferation, function, antitumor immunity, and memory. F2 gel provides a danger signal via the innate immune system, and at the same time serves as a sustained release delivery vehicle for Ag and IL-12 at the vaccine site. This creates a potent microenvironment so that Ag is presented in association with a danger signal, whereas IL-12 enhances activation of APCs and IFN-{gamma} production. Systemic toxicity is currently a major limitation to the use of IL-12 in human clinical trials. However, the lower serum levels of IL-12 associated with paracrine administration are likely to be associated with a significant reduction in the toxicity associated with systemic administration of IL-12. The relatively simple and inexpensive production of F2 gel, as well as its efficacy and potential for significant reduction in toxicity, provide rationale for the clinical translation of this approach.


   Footnotes
 
1 This work was supported by the National Institutes of Health Grant 1 R01 CA94856-01, and a grant from Marine Polymer Technologies (Danvers, MA). Back

2 Address correspondence and reprint requests to Dr. William E. Gillanders, Department of Surgery, Medical University of South Carolina, 96 Jonathan Lucas Street, PO Box 250613, Charleston, SC 29425. E-mail address: gillanwe{at}musc.edu Back

Received for publication March 27, 2003. Accepted for publication February 9, 2004.


   References
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 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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Alum with Interleukin-12 Augments Immunity to a Melanoma Peptide Vaccine: Correlation with Time to Relapse in Patients with Resected High-Risk Disease
Clin. Cancer Res., January 1, 2007; 13(1): 215 - 222.
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