- Email: [email protected]
High-avidity antitumor T-cell generation by toll receptor 8–primed, myeloid- derived dendritic cells is mediated by IL-12 production
High-avidity antitumor T-cell generation by toll receptor 8 –primed, myeloid- derived dendritic cells is mediated by IL-12 production Shuwen Xu, MD,a Ursula Koldovsky, PhD,a Min Xu, MD,a Daniel Wang,a Elizabeth Fitzpatrick,a Gilsoo Son, MD,a Gary Koski, PhD,b and Brian J. Czerniecki, MD, PhD,a Philadelphia, Penn, and Cleveland, Ohio
Background. High-level production of heterodimeric p70 interleukin (IL)-12 by myeloid-derived dendritic cells (DCs) requires 2 signals: interferon gamma (IFN-␥) and a maturation signal provided by CD40 ligation (CD40L) or lipopolysaccharide (LPS). Methods. In the current study we demonstrate that signaling through toll-like receptor (TLR) 8, but not TLR3, TLR2, or TLR4, provides a priming signal to myeloid-derived DC for high IL-12 p70 heterodimer production. Results. All the TLR agonists induced maturation of DC as evidenced by increased expression of CD83, CD80, and CD86. Both IFN-␥ and TLR7/8 agonist R848 increased expression of TLR8 in immature monocyte-derived DCs. The combination of TLR7/8 agonist R848 and maturation signals LPS or CD40L induced high-level expression of IL-12p35 and p40 similar to that induced by IFN-␥ plus LPS. In contrast, receptor agonists specific for TLR7 did not prime for IL-12 production. The p70 IL-12 produced by the TLR8-primed DC polarized CD4⫹ T for Th1 cytokine production and induced CD8⫹ T cells, displaying high functional avidity with enhanced tumor cell recognition. Conclusions. The data suggest that toll 8 receptor agonists are useful for inducing type-1 polarized DCs for vaccine design in treating cancer and infectious disease. (Surgery 2006;140:170-8.) From the Harrison Department of Surgical Research and the Department of Surgery, University of Pennsylvania,a and the Center for Surgical Research, Cleveland Clinicb
Functional interleukin (IL)-12 (p70) is a heterodimer composed of heavy (p40) and light (p35) chains. It generally is associated with polarizing Th1-type immune responses necessary for resisting intracellular pathogens. Production of IL-12 p70 by human dendritic cells (DCs) requires 2 signals.1 The signals required include a priming signal supplied by interferon (IFN)-␥ and a maturation signal
Presented at the 67th Annual Meeting of the Society of University Surgeons, First Annual Academic Surgical Congress, February 7-11, 2006, San Diego, California. Supported by American Cancer Society grant RSG-99-029-04LIB and National Institutes of Health grant RO1-CA-096997-02. Accepted for publication March 10, 2006. Reprint requests: Brian J. Czerniecki, MD, PhD, Dept of Surgery, 4 Silverstein, 3400 Spruce St, Philadelphia, PA 19104. E-mail: [email protected]. 0039-6060/$ - see front matter © 2006 Mosby, Inc. All rights reserved. doi:10.1016/j.surg.2006.03.006
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provided by CD40 ligand (CD40L) or lipopolysaccharide (LPS). It has been demonstrated that both IFN-␥ and LPS are necessary for p35 chain expression2 and that a lack of p35 induction can lead to the production of p40 homodimers, which have been shown to inhibit Th1 signaling and enhance Th2 function.3 We have demonstrated that high level IL-12 production by DCs primes CD4 T cells for secretion of Th1 cytokines.4 In contrast, IL-12–secreting DCs do not strongly polarize cytokine secretion by CD8⫹ T cells, but instead greatly enhance the T cells’ functional avidity (ie, antigen sensitivity) probably in part through increased expression of CD8 ␣.4 This enhanced functional avidity endowed tumor antigen–sensitized T cells with a potent capacity to recognize and kill tumor cells. T cells demonstrating high functional avidity have been suggested to be essential for effective T-cell therapy for treating patients with cancer.5 Given these associations between IL-12 p70 production, enhanced T-cell functional avidity, and the heightened capacity to
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recognize and kill tumor cells, the demonstration of other novel pathways for induction of IL-12 in DCs could prove useful in the development of new and improved adjuvants for use in cancer immunotherapy. Recent work from our laboratory has demonstrated that single-stranded RNA (ssRNA) with features characteristic of bacterial pathogens can replace IFN-␥ and prime DC for high production of IL-126 upon maturation. Since toll receptors 7 and 8 have been shown to bind ssRNA,7,8 and others9,10 have demonstrated that toll-like receptor 8 (TLR8) agonists can induce IL-12 production in murine- and human myeloid-derived DCs, we sought to determine whether activation of TLR8 could prime myeloid DCs activated in a rapid serum-free system for high production of IL-12. In addition, we explored whether IL-12 produced by TLR8-primed DCs was responsible for inducing antitumor CD8⫹ T cells similar to DCs primed by IFN-␥.4,11 The current study demonstrates that TLR8 signaling primes myeloidderived DCs for high production of IL-12 p70 and that the IL-12 produced by TLR8 priming is responsible for inducing CD8⫹ T cells that exhibit increased functional avidity for tumor cells. Such type 1 polarized DCs primed by TLR8 agonist resimiquod (R848) has important implications not only for developing DC vaccines for cancer therapy, but also for the development of TLR8 agonists as potent anticancer adjuvants. MATERIAL AND METHODS Preparation of fractions of human peripheral blood mononuclear cells. Healthy donors provided informed consent and underwent leukapheresis. Blood products were then elutriated and cryopreserved as described.12 Reagents and antibodies. Fluorescein isothiocyanate (FITC)- or phycoerythrin-conjugated mouse antihuman CD14, CD80, CD86, and CD83 were from BD Biosciences (San Diego, Calif). FITC- or PE-conjugated goat antihuman TLR2, TLR3, TLR4, and TLR8 were from Imgenex (San Diego, Calif). Lipoteichoic acid (LTA), polyinosinic-polycytyidilyc acid (poly [I: C]) (Invivogen), E.coli O26:B6 LPS (Sigma-Aldrich, St. Louis, Mo), R848 (a kind gift from 3M pharmaceuticals), and CD40L (a kind gift from Amgen) were used for activation of DCs. Generation of monocyte-derived dendritic cells. Monocytes were plated (1.5 million/mL) in macrophage serum-free medium (Life Technologies) in the presence of 50 ng/mL granulocyte-macrophage colony-stimulating factor (GM-CSF). After over-
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night culture, the cells were treated either with 2 g/mL LTA, 50 g/mL poly (I: C),10 ng/mL LPS, and 1g/mL R848 alone for 24 hours or primed first with 1,000 /mL IFN-␥, LTA, poly (I: C), and R848, respectively, for 2 hours, then matured with LPS or 1 g/mL CD40L for an additional 20 hours. For comparison, cytokine maturation mixture (CMM), the “gold standard” according to the formulation of Jonuleit,13 was used to induce DCs. CD4ⴙ and CD8ⴙ T-cell sensitizations. Lymphocyte-rich elutriation fractions (120 and 140 cc/ min) were used to prepare either CD4⫹ T cells or CD8⫹ cells with the use of negative depletion columns according to the manufacturers’ recommendations (R&D) as described previously.4 Purified allogeneic CD4⫹/CD45RA⫹ T cells (1 ⫻ 106/well) as described above were cocultured with DCs (1 ⫻ 105/well) as described above in 48-well tissue culture plates. On day 6 the T cells were harvested and restimulated on plates coated with anti-CD3 and anti-CD28 as described previously.4 Supernatants were harvested 24 hours later and analyzed by enzyme-linked immunosorbent assay (ELISA). For in vitro sensitization of CD8⫹ T cells, DCs from HLAA2⫹ normal donors were pulsed with MART-1 (2735) peptides at 10 ng/mL 2 hours before harvest. Harvested cells were washed twice and plated in fresh Iscove medium, 5% human AB serum, and 30 IU IL-2 with purified CD8⫹ cells at a T cell to DC ratio of 20:1. After 1 week the T cells were harvested and restimulated with relevant, antigen-expressing melanoma tumor cells (MEL 624 HLAA2⫹). Also tested were negative control (MEL 624 HLA-A2⫺ and MW115, HLA-A2⫹ and tumor antigen⫺) cell lines that express either MART-1 antigen or HLA-A2 antigen, but not both. CD8⫹ T cells also were tested against HLA-A2 transporter– deficient T2 cells pulsed with relevant and irrelevant peptides. Supernatants were harvested after 24 hours and analyzed by ELISA. Northern blot analysis and probes. Monocytes were activated as described above. Cells were then lysed, and RNA was analyzed on Northern blots as described.14 The probe for human TLR8 was purified complementary DNA derived from a plasmid (a kind gift from Dr Bruce Beutler, The Scripps Research Institute, La Jolla, Calif). Probes for human IL-12p35, p40 were excised from the plasmids obtained from Open Biosystems (Huntsville, Ala). RESULTS Human monocytes constitutively express TLR2, TLR3, TLR4, and TLR8. TLR agonists have been demonstrated to activate monocyte-derived DCs and stimulate innate immunity.15 One must under-
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Fig 1. Expression of TLR2, TLR3, TLR4, and TLR8 in human monocytes. Monocyte surface expression of TLR2, TLR3, TLR4 and TLR8 (A) and intracellular expressions of TLR2, TLR3, TLR4, and TLR8 (B) were assessed by flow cytometry. Shaded histogram represents isotype-matched negative control Ab staining; open histogram represents a specific Ab staining. Results are representative of 4 experiments with different donors. TLR, Toll-like receptor.
stand TLR expression patterns in monocytes to study the effects of TLR agonists on DCs. Thus, our experiments were initiated to assess surface and intracellular TLR expressions of human monocytes. By flow cytometric analysis, we found that monocytes expressed surface TLR2 and TLR4, but not TLR3 and TLR8 (Fig 1, A). However, intracellular staining detected expression of not only TLR2 and TLR4, but also TLR3 and TLR8 (Fig 1, B), indicating TLR3 and TLR8 reside only intracellularly. There was no evidence of TLR7 or TLR9 expression (not shown). IFN-␥ and R848 upregulate TLR8 expression in monocyte-derived DCs. TLR8 signaling has been shown to synergize with IFN-␥ to activate DCs. To study the mechanism of its interaction with IFN-␥ or other possible TLR agonists, we examined regulation of expression of TLR8 messenger RNA (mRNA) in monocyte-derived DCs. We stimulated immature DCs with IFN-␥ or TLR agonists, respectively. On the basis of the human monocytes’ expression of TLR2, TLR3, TLR4, and TLR8, their corresponding ligand LTA, poly (I: C), LPS and R848 were used in the studies. DCs treated with IFN-␥ or R848 for 4 hours expressed large amounts (4- to 5-fold increase) of TLR8 mRNA, and LPS moderately upregulated TLR8 mRNA, whereas LTA and poly(I: C) had no effect on TLR8 expression (Fig 2). TLR agonists induce maturation and expression of DC costimulatory molecules. Because alteration in surface protein expression is an important step
in the maturation of DCs, we next investigated whether TLR agonists had an additive effect on expression of DC costimulatory molecules. Immature DCs were activated either with a single TLR agonist for 24 hours or primed first with a TLR agonist for 2 hours, then matured with LPS for an additional 20 to 24 hours. The surface phenotype was then analyzed by flow cytometry. DCs activated by a single TLR signal (Fig 3, A) displayed levels of costimulatory molecules comparable to those expressed on DCs induced by 2 TLR signals (Fig 3, B). All DCs showed similar low levels of CD14, and upregulated CD80, CD86, and CD83. This finding implies that single TLR signaling possesses the capacity to fully mature DCs, but contemporaneous signaling through additional TLR family members has little additional effect on expression of DC costimulatory molecules. TLR8 agonist R848 primes DCs for high level of IL-12 production. High levels of IL-12 secretion in human DCs require 2 signals,1 such as an initial priming signal supplied by IFN-␥ and a maturation signal provided by LPS or CD40L. The importance of IL-12 in tumor immunity prompted us to explore whether other signaling molecules could substitute for IFN-␥ in DC priming. Since TLR agonists have been shown to induce Th1 immunity, we therefore examined their capacity to prime DCs for IL-12 production. Immature DCs were primed with a single TLR agonist LTA, poly (I: C), LPS, or R848 separately for 2 hours and then matured with either LPS or CD40L for 16 to 20 hours. Culture
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Fig 2. TLR8 mRNA expression in human monocyte– derived immature DCs is regulated positively by IFN-␥ and R848. Overnight-cultured human monocytes (immature DCs) were treated with IFN-␥, LTA, poly (I: C), LPS, or R848, respectively, for 4 hours. Cells were harvested, and RNAs were extracted for analysis of expressions of TLR8 and GAPDH by Northern blots. Results are representative of 5 experiments. GAPDH, Glyceraldehyde-3-phosphate dehydrogenase; GM-CSF, granulocyte-macrophage colony-stimulating factor; IFN-␥, interferon gamma; LPS, lipopolysaccharide; LTA, lipoteichoic acid; TLR, toll-like receptor; poly (I: C), polyinosinic-polycytyidilyc acid.
Fig 3. DCs express comparable levels of costimulatory molecules when induced by either a single TLR signaling or multiple TLR agonists. Human monocytes were cultured in serum-free medium plus GM-CSF overnight (immature DCs); they were then treated with LTA, poly (I: C), LPS, or R848, respectively, for 24 hours (A); immature DCs were primed with LTA, poly (I: C), and R848 separately for 2 hours, then further matured with LPS for additional 20-24 hours (B). DCs were harvested and surface phenotype was analyzed by flow cytometry. For CD14, filled traces represent CD14 staining of monocyte controls (starting populations), and open traces represent CD14 staining of cultured DCs. For CD80, CD86 and CD83, filled traces represent isotype-matched negative control staining; open traces represent specific Ab staining. Results are representative of 5 experiments. GM/IL-4, granulocyte-macrophage colony-stimulating factor/ interleukin-4; IFN-␥, interferon gamma; IL-12, interleukin 12; LPS, lipopolysaccharide; LTA, lipoteichoic acid; poly (I: C), polyinosinic-polycytyidilyc acid; R848, resimiquod.
supernatants were harvested for measurements of IL-12p70 by ELISA. For comparison, we also treated immature DCs with TLR agonists alone for 24 hours or primed the DCs with IFN-␥ for 2 hours,
then matured the cells with TLR agonists, respectively, for 20 hours. Figure 4, A demonstrates that DCs induced by a single TLR agonist secreted no IL-12p70; DCs primed with IFN-␥ and then ma-
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Fig 4. TLR8 agonist R848 primes DCs for high level of IL-12 production. Immature DCs were treated with lipotechoic acid (LTA), poly (I: C), LPS, or R848 individually for 2 hours and then stimulated with either LPS (black bar) or CD40L (dotted bar) for 20 hours. For comparison, immature DCs were activated by LTA, poly (I: C), LPS, or R848 alone for 24 hours (white bar), or primed with IFN-␥ first for 2 hours and then stimulated with LTA, poly (I: C), LPS, or R848, respectively, for 20 hours (horizontal line bar). IL-12p70 secretions in culture supernatants were measured by ELISA (A). In separate experiments, cells were treated with a single TLR agonist for 4 hours or primed with TLR agonists individually for 2 hours and further activated by LPS for additional 2 hour. Total RNA was extracted for IL-12p35 and p40 expression analysis by Northern blot (B and C). Results are representative of 3 experiments. GAPDH, Glyceraldehyde-3-phosphate dehydrogenase; GM-CSF, granulocyte-macrophage colony-stimulating factor; IFN-␥, interferon gamma; IL-12, interleukin 12; LPS, lipopolysaccharide; LTA, lipoteichoic acid; poly (I: C), polyinosinic-polycytyidilyc acid; R848, resimiquod.
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tured with a TLR agonist produced high levels of IL-12p70 as expected. LTA, poly (I: C), and LPS, which trigger TLR2, TLR3, and TLR4, were unable to prime DCs for IL-12p70 production; only the TLR8 agonist R848 displayed the unique property of priming DC for high IL-12p70 production. In a parallel experiment we also examined the expression pattern of IL-12p35 and p40 mRNA. DCs activated by a single TLR agonist induced only IL12p40 mRNA transcript but no p35 (Fig 4, B); DCs primed with LTA or poly (I: C) and subsequently matured with LPS failed to induce IL-12p35 (Fig 4, C). In a separate experiment DCs primed with TLR7 agonist 7-Deaza-2’deoxyguanosine and matured with LPS also were unable to express IL12p35 mRNA (data not shown). In contrast, DCs primed with IFN-␥ or R848 followed by LPS maturation expressed both IL-12p35 and p40 genes (Fig 4, C). These findings suggest that induction of the IL-12 p35 gene requires 2 signals: Only TLR8 agonist R848 could replace IFN-␥ as a priming signal, synergizing with LPS or CD40L in the production of DC IL-12p70. R848 and LPS synergistically induce TNF-␣ and IL-6 secretion by DCs. We next tested whether other cytokines were induced by the TLR agonists. TNF-␣ and IL-6, 2 Th1 cytokines, also were induced synergistically by a combined treatment of R848 and LPS as shown in Figure 5. Interestingly unlike IFN-␥, R848 treatment of immature DCs stimulated significant IL-6 production (Fig 5). In addition, although poly (I: C) and LPS did not induce IL-12 production, there was evidence of production of both IL-6 and TNF-␣ (Fig 5). R848-primed DCs display Th1-polarizing capacity. To further study the functional ability of R848-primed DCs, we carried out allosensitization experiments to analyze their capacity to functionally polarize CD4⫹ T cells. Naïve allogeneic CD4⫹T cells were stimulated by CMM-induced DCs12 or DCs primed with R848, IFN-␥, or poly (I: C), respectively. Their functional phenotype was assessed by ELISA of culture supernatants. R848- or IFN-␥–primed DCs, which secreted high levels of IL-12, stimulated naïve CD4⫹T cells to produce large quantities of IFN-␥ with little IL-4, whereas poly(I: C) primed–DCs or CMM-DCs, which produced little or no IL-12, stimulated T cells to secrete much less IFN-␥, but abundant IL-4 (Fig 6,A). Thus, these 2 distinct types of DCs polarized CD4⫹ T cells into being characteristic of either the Th1 or Th2 phenotype. The Th1-polarized CD4⫹T cells induced by R848-primed DCs could be blocked by IL-12– neutralizing monoclonal antibody (Fig 6, B).
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Fig 5. R848 and LPS synergistically induce TNF-␣ and IL-6 secretion by DCs. Immature DCs were treated with IFN-␥, LTA, poly (I:C), LPS, or R848, respectively, for 24 hours (white bar), or they were primed with IFN-␥ LTA, poly (I: C), or R848 for 2 hours, followed by LPS treatment for 20 hours (black bar). Culture supernatants were collected for cytokine assays by ELISA. Results are representative of 5 experiments with different donors. IFN-␥, Interferon gamma; IL-12, interleukin 12; LPS, lipopolysaccharide; LTA, lipoteichoic acid; poly (I: C), polyinosinic-polycytyidilyc acid; R848, resimiquod; TNF-␣, tumor necrosis factor ␣.
These results demonstrate that IL-12–producing DCs primed by R848 have a high capacity to polarize CD4⫹ T cells toward a Th1 phenotype. R848-primed DCs confer CD8ⴙ T cells with an enhanced ability to recognize tumor cells. We have shown previously that IL-12–producing DCs generate high functional avidity CD8⫹ T cells, which have an enhanced ability to recognize tumor cells. We therefore tested whether R848-primed DCs also displayed this property. As Figure 7, A and C demonstrate, poly(I: C)–primed DCs or CMM-DCs, which produced little or no IL-12–induced CD8⫹ T cells, recognizing only T2 target cells pulsed with MART-1 (27-35), the peptide to which they were sensitized, but not tumor cells expressing MART-1. In contrast, IL-12–producing DCs primed by either R848 or IFN-␥ sensitized CD8⫹ T cells for large quantities of Ag-specific IFN-␥ release in response to both T2 cells pulsed with MART-1 (27-35) and melanoma cells positive for HLA-A2, which ex-
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Fig 6. R848-primed DCs display Th1-polarizing capacity. Naïve allogeneic CD4⫹ T cells were stimulated by CMMinduced DCs or DCs primed with R848, IFN-␥, or poly (I: C), respectively, and matured with CD40L. On day 6, activated T cells were restimulated with plate-bound anti-CD3 and anti-CD28 for 24 hours. A, Supernatants were analyzed for cytokine secretion by ELISA. B, ELISA analysis of supernatants from restimulated T cells cocultured with DCs generated by R848/CD40L in the presence of IL-12–neutralizing Ab or isotype-matched control mucosal antibody. Results are representative of 3 experiments. Ab, antibody; CMM-DC, cytokine maturation mixture-dendritic cell; IFN-␥, interferon gamma; IL-12, interleukin 12; LPS, lipopolysaccharide; LTA, lipoteichoic acid; poly (I: C), polyinosinic-polycytyidilyc acid; R848, resimiquod.
pressed endogenous melanoma antigen MART-1. IL-12–neutralizing antibody blocked such tumor recognition. However, these T cells still recognized target T2 cells pulsed with the relevant peptide (Fig 7, B and D). Again, these results demonstrate the central role of IL-12 in tumor recognition by CD8⫹ T cells. DISCUSSION Mature DCs secreting high levels of IL-12p70 have the capacity to sensitize CD8⫹ T cells to tumor-associated antigens and endow them with enhanced antigen sensitivity (ie, functional avidity).4 Indeed, we demonstrated recently that this increase in functional avidity of CD8⫹ T cells is critical for direct recognition and destruction of tumor cells. These recent findings regarding the importance of IL-12 p70 secretion by DCs and the subse-
quent induction of T cells with high functional avidity already is serving as the basis for an ongoing clinical vaccine trial against ductal carcinoma in situ (DCIS). Here, IL-12 p70 –secreting DCs activated with IFN-␥ and LPS are pulsed with HER-2/ neu peptides and then administered intranodally to subjects with this carcinoma. These DCs are sensitizing high functional avidity anti–HER-2/neu T cells in vivo that are inducing apparent clinical responses (manuscript in preparation). To date IFN-␥, ssRNA, or contact with natural killer cells have been the principal ways to prime DCs for high secretion of IL-12 p70 upon subsequent maturation.6,10 TLR agonists have been shown to mature DC, and some paired combinations of TLR agonists (but not others) can induce DCs to secrete large amounts of IL-12 p70 as well.16,17 We demonstrate here that the synthetic
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Fig 7. R848 primes DCs for high level of IL-12 production, accounting for enhanced tumor recognition by sensitized CD8⫹ T cells. Immature DCs were primed with IFN-␥, R-848, or poly (I: C), respectively, for 2 hours and further matured with CD40L for 20 hours. They were pulsed with peptide MART-1 (27-35) 2 hours before harvest. DCs were then cocultured with purified CD8⫹ T cells. After 7 days, T cells were harvested and tested for reactivity (IFN-␥ release) to target melanoma cell lines or to T2 cells pulsed with relevant (MART-1 [27-35]) and irrelevant peptides (A and C) by ELISA analysis of 24-hour culture supernatants. Adding neutralizing anti–IL-12 to the coculture of T cells and DCs blocked the tumor recognition of CD8⫹ T cells induced by R848-primed and CD40L-matured DCs. However, these T cells still recognized target T2 cells pulsed with optimal level of MART-1 (27-35) peptide (B and D). Results are representative of 4 experiments from different donors. Ab, antibody; CD40L, CD40 ligation; CMM-DC, cytokine maturation mixture-dendritic cell; IFN-␥, interferon gamma; IL-12, interleukin 12; poly (I: C), polyinosinic-polycytyidilyc acid; R848, resimiquod
TLR8 agonist R848 primes myeloid-derived DCs for high-level production of IL-12 upon subsequent maturation with LPS or CD40L. Our results agree with those of Gautier et al16 as well as Napolitani et al,17 demonstrating that multiple TLR agonists can stimulate IL-12 production by DCs. However, the mechanism by which TLRs prime DCs for high-level production of IL-12 is not entirely clear. It has been suggested that type-I interferons induced by TLRs provide priming signals in the DCs,15 but not all studies have observed evidence of this effect.16 In that regard, the TLR3 agonist poly (I: C) used in this study induces type-I interferon in these DCs,6 but in amounts insufficient to prime DCs for IL-12 production. We also made the interesting observation that both IFN-␥ and R848 increased dramatically the
levels of TLR8 expression in the DCs. Because each of these signals can cooperate, both with each other and individually in combination with other TLR agonists, to induce high-level secretion of IL12, possible biologic relationships between their respective signaling pathways are suggested. Indeed, preliminary data from our laboratory suggest that both IFN-␥ and R848 can greatly increase the expression of both interferon regulatory factors 1 and 8, 2 nuclear factors that are critical for the regulation of IL-12 expression.18 But what are the possible roles of TLR agonists in improving vaccines against malignancies and infectious agents? Natural ligands for TLRs often are complex biopolymers that may be difficult to purify in sufficient quantity from cultured organisms, relatively expensive to synthesize, and sometimes easily de-
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graded. For example, whereas our studies show particular promise for TLR8 agonists in priming DCs for IL-12 secretion and subsequent sensitization of high functional avidity, tumor-recognizing CD8⫹ T cells, the natural ligand, ssRNA, is broken down quickly by nucleases. Fortunately, small organic molecules such as R848 can make an excellent substitute, and other small organic pharmaceuticals that exploit the capacity to activate various TLRs undoubtedly are on the way. Identification of pathways leading to IL-12 production may provide additional insights into design and use of such new adjuvants for treating cancer and infectious diseases that require Th 1–polarized T cells. REFERENCES 1. Snijders A, Kalinski P, Hilkens CM, Kapsenberg ML. Highlevel IL-12 production by human dendritic cells requires two signals. Int Immunol 1998;10(11):1593-8. 2. Liu, J, Cao S, Herman LM, Ma X. Differential regulation of interleukin (IL)-12 p35 and p40 gene expression and interferon (IFN)-{gamma}-primed IL-12 production by IFN regulatory factor 1. J Exp Med 2003;198(8):1265-76. 3. Kalinski P, Vieira P, Schuitemaker JH, de Jong EC, Kapsenberg ML. Prostaglandin E2 is a selective inducer of interleukin-12 p40 (IL-12p40) production and an inhibitor of bioactive IL-12p70 heterodimer. Blood 2001;97(11):3466-9. 4. Xu S, Koski G, Faries M, et al. Rapid high efficiency sensitization of CD8⫹ T cells to tumor antigens by dendritic cells leads to enhanced functional avidity and direct tumor recognition through an IL-12-dependent mechanism. J Immunol 2003; 171(5):2251-61. 5. Dutoit V, Rubio-Godoy V, Dietrich PY, et al. Heterogeneous T-cell response to MAGE-A10254-262: high avidity-specific cytolytic T lymphocytes show superior antitumor activity. Cancer Res 2001;61(15):5850-6. 6. Koski GK, Kariko K, Xu S, Weissman D, Cohen PA, Czerniecki BJ. Cutting edge: innate immune system discriminates between RNA containing bacterial versus eukaryotic structural features that prime for high-level IL-12 secre-
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tion by dendritic cells. J Immunol 2004;172(7): 3989-93. Heil F, Hemmi H, Hochrein H, et al. Species-specific recognition of single-stranded RNA via toll-like receptor 7 and 8 Science 2004;303(5663): p. 1526-1529. Diebold SS, Kaisho T, Hemmi H, et al. Innate antiviral responses by means of TLR7-mediated recognition of singlestranded RNA. Science 2004;303(5663):1529-31. Hemmi H, Kaisho T, Takeuchi O, et al. Small anti-viral compounds activate immune cells via the TLR7 MyD88dependent signaling pathway. Nat Immunol 2002; 3(2):196-200. Gorden KB, Gorski KS, Gibson SJ, et al. Synthetic TLR agonists reveal functional differences between human TLR7 and TLR8. J Immunol 2005;174(3):1259-68. Kalinski P, Giermasz A, Nakamura Y, et al. Helper role of NK cells during the induction of anticancer responses by dendritic cells. Mol Immunol 2005 42(4):535-9. Czerniecki BJ, Carter C, Rivoltini L, et al. Calcium ionophore-treated peripheral blood monocytes and dendritic cells rapidly display characteristics of activated dendritic cells. J Immunol 1997;59(8):3823-37. Jonuleit H, Giesecke-Tuettenberg A, Tuting T, et al. A comparison of two types of dendritic cell as adjuvants for the induction of melanoma-specific T cell responses in humans following intranodal injection. Int J Cancer 2001 93:-243. Kariko K, Ni HP, Capodici J, Lamphier M, Weissman D, et al. mRNA is an endogenous ligand for toll-like teceptor 3. J Biol Chem 2004;279(13):12542-50. Medzhitov R. Toll-like receptors and innate immunity. Nat Rev Immunol 2001;(2):135-45. Gautier G, Humbert M, Deauvieau F, et al. A type I interferon autocrine-paracrine loop is involved in toll-like receptor-induced interleukin-12p70 secretion by dendritic cells. J Exp Med 2005;201(9):1435-46. Napolitani G, Rinaldi A, Bertoni F, Sallusto F, Lanzavecchia A, et al. Selected toll-like receptor agonist combination synergistically trigger a T helper type 1-polarizing program in dendritic cells. Nat Immunol 2005;6(8):769-76. Liu J, Guan X, Tamura T, Ozato K, Ma X. Synergistic activation of interleukin-12 p35 gene transcription by interferon regulatory factor-1 and interferon consensus sequence-binding protein. J Biol Chem 2004;279(53): 55609-17.
Background. High-level production of heterodimeric p70 interleukin (IL)-12 by myeloid-derived dendritic cells (DCs) requires 2 signals: interferon gamma (IFN-␥) and a maturation signal provided by CD40 ligation (CD40L) or lipopolysaccharide (LPS). Methods. In the current study we demonstrate that signaling through toll-like receptor (TLR) 8, but not TLR3, TLR2, or TLR4, provides a priming signal to myeloid-derived DC for high IL-12 p70 heterodimer production. Results. All the TLR agonists induced maturation of DC as evidenced by increased expression of CD83, CD80, and CD86. Both IFN-␥ and TLR7/8 agonist R848 increased expression of TLR8 in immature monocyte-derived DCs. The combination of TLR7/8 agonist R848 and maturation signals LPS or CD40L induced high-level expression of IL-12p35 and p40 similar to that induced by IFN-␥ plus LPS. In contrast, receptor agonists specific for TLR7 did not prime for IL-12 production. The p70 IL-12 produced by the TLR8-primed DC polarized CD4⫹ T for Th1 cytokine production and induced CD8⫹ T cells, displaying high functional avidity with enhanced tumor cell recognition. Conclusions. The data suggest that toll 8 receptor agonists are useful for inducing type-1 polarized DCs for vaccine design in treating cancer and infectious disease. (Surgery 2006;140:170-8.) From the Harrison Department of Surgical Research and the Department of Surgery, University of Pennsylvania,a and the Center for Surgical Research, Cleveland Clinicb
Functional interleukin (IL)-12 (p70) is a heterodimer composed of heavy (p40) and light (p35) chains. It generally is associated with polarizing Th1-type immune responses necessary for resisting intracellular pathogens. Production of IL-12 p70 by human dendritic cells (DCs) requires 2 signals.1 The signals required include a priming signal supplied by interferon (IFN)-␥ and a maturation signal
Presented at the 67th Annual Meeting of the Society of University Surgeons, First Annual Academic Surgical Congress, February 7-11, 2006, San Diego, California. Supported by American Cancer Society grant RSG-99-029-04LIB and National Institutes of Health grant RO1-CA-096997-02. Accepted for publication March 10, 2006. Reprint requests: Brian J. Czerniecki, MD, PhD, Dept of Surgery, 4 Silverstein, 3400 Spruce St, Philadelphia, PA 19104. E-mail: [email protected]. 0039-6060/$ - see front matter © 2006 Mosby, Inc. All rights reserved. doi:10.1016/j.surg.2006.03.006
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provided by CD40 ligand (CD40L) or lipopolysaccharide (LPS). It has been demonstrated that both IFN-␥ and LPS are necessary for p35 chain expression2 and that a lack of p35 induction can lead to the production of p40 homodimers, which have been shown to inhibit Th1 signaling and enhance Th2 function.3 We have demonstrated that high level IL-12 production by DCs primes CD4 T cells for secretion of Th1 cytokines.4 In contrast, IL-12–secreting DCs do not strongly polarize cytokine secretion by CD8⫹ T cells, but instead greatly enhance the T cells’ functional avidity (ie, antigen sensitivity) probably in part through increased expression of CD8 ␣.4 This enhanced functional avidity endowed tumor antigen–sensitized T cells with a potent capacity to recognize and kill tumor cells. T cells demonstrating high functional avidity have been suggested to be essential for effective T-cell therapy for treating patients with cancer.5 Given these associations between IL-12 p70 production, enhanced T-cell functional avidity, and the heightened capacity to
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recognize and kill tumor cells, the demonstration of other novel pathways for induction of IL-12 in DCs could prove useful in the development of new and improved adjuvants for use in cancer immunotherapy. Recent work from our laboratory has demonstrated that single-stranded RNA (ssRNA) with features characteristic of bacterial pathogens can replace IFN-␥ and prime DC for high production of IL-126 upon maturation. Since toll receptors 7 and 8 have been shown to bind ssRNA,7,8 and others9,10 have demonstrated that toll-like receptor 8 (TLR8) agonists can induce IL-12 production in murine- and human myeloid-derived DCs, we sought to determine whether activation of TLR8 could prime myeloid DCs activated in a rapid serum-free system for high production of IL-12. In addition, we explored whether IL-12 produced by TLR8-primed DCs was responsible for inducing antitumor CD8⫹ T cells similar to DCs primed by IFN-␥.4,11 The current study demonstrates that TLR8 signaling primes myeloidderived DCs for high production of IL-12 p70 and that the IL-12 produced by TLR8 priming is responsible for inducing CD8⫹ T cells that exhibit increased functional avidity for tumor cells. Such type 1 polarized DCs primed by TLR8 agonist resimiquod (R848) has important implications not only for developing DC vaccines for cancer therapy, but also for the development of TLR8 agonists as potent anticancer adjuvants. MATERIAL AND METHODS Preparation of fractions of human peripheral blood mononuclear cells. Healthy donors provided informed consent and underwent leukapheresis. Blood products were then elutriated and cryopreserved as described.12 Reagents and antibodies. Fluorescein isothiocyanate (FITC)- or phycoerythrin-conjugated mouse antihuman CD14, CD80, CD86, and CD83 were from BD Biosciences (San Diego, Calif). FITC- or PE-conjugated goat antihuman TLR2, TLR3, TLR4, and TLR8 were from Imgenex (San Diego, Calif). Lipoteichoic acid (LTA), polyinosinic-polycytyidilyc acid (poly [I: C]) (Invivogen), E.coli O26:B6 LPS (Sigma-Aldrich, St. Louis, Mo), R848 (a kind gift from 3M pharmaceuticals), and CD40L (a kind gift from Amgen) were used for activation of DCs. Generation of monocyte-derived dendritic cells. Monocytes were plated (1.5 million/mL) in macrophage serum-free medium (Life Technologies) in the presence of 50 ng/mL granulocyte-macrophage colony-stimulating factor (GM-CSF). After over-
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night culture, the cells were treated either with 2 g/mL LTA, 50 g/mL poly (I: C),10 ng/mL LPS, and 1g/mL R848 alone for 24 hours or primed first with 1,000 /mL IFN-␥, LTA, poly (I: C), and R848, respectively, for 2 hours, then matured with LPS or 1 g/mL CD40L for an additional 20 hours. For comparison, cytokine maturation mixture (CMM), the “gold standard” according to the formulation of Jonuleit,13 was used to induce DCs. CD4ⴙ and CD8ⴙ T-cell sensitizations. Lymphocyte-rich elutriation fractions (120 and 140 cc/ min) were used to prepare either CD4⫹ T cells or CD8⫹ cells with the use of negative depletion columns according to the manufacturers’ recommendations (R&D) as described previously.4 Purified allogeneic CD4⫹/CD45RA⫹ T cells (1 ⫻ 106/well) as described above were cocultured with DCs (1 ⫻ 105/well) as described above in 48-well tissue culture plates. On day 6 the T cells were harvested and restimulated on plates coated with anti-CD3 and anti-CD28 as described previously.4 Supernatants were harvested 24 hours later and analyzed by enzyme-linked immunosorbent assay (ELISA). For in vitro sensitization of CD8⫹ T cells, DCs from HLAA2⫹ normal donors were pulsed with MART-1 (2735) peptides at 10 ng/mL 2 hours before harvest. Harvested cells were washed twice and plated in fresh Iscove medium, 5% human AB serum, and 30 IU IL-2 with purified CD8⫹ cells at a T cell to DC ratio of 20:1. After 1 week the T cells were harvested and restimulated with relevant, antigen-expressing melanoma tumor cells (MEL 624 HLAA2⫹). Also tested were negative control (MEL 624 HLA-A2⫺ and MW115, HLA-A2⫹ and tumor antigen⫺) cell lines that express either MART-1 antigen or HLA-A2 antigen, but not both. CD8⫹ T cells also were tested against HLA-A2 transporter– deficient T2 cells pulsed with relevant and irrelevant peptides. Supernatants were harvested after 24 hours and analyzed by ELISA. Northern blot analysis and probes. Monocytes were activated as described above. Cells were then lysed, and RNA was analyzed on Northern blots as described.14 The probe for human TLR8 was purified complementary DNA derived from a plasmid (a kind gift from Dr Bruce Beutler, The Scripps Research Institute, La Jolla, Calif). Probes for human IL-12p35, p40 were excised from the plasmids obtained from Open Biosystems (Huntsville, Ala). RESULTS Human monocytes constitutively express TLR2, TLR3, TLR4, and TLR8. TLR agonists have been demonstrated to activate monocyte-derived DCs and stimulate innate immunity.15 One must under-
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Fig 1. Expression of TLR2, TLR3, TLR4, and TLR8 in human monocytes. Monocyte surface expression of TLR2, TLR3, TLR4 and TLR8 (A) and intracellular expressions of TLR2, TLR3, TLR4, and TLR8 (B) were assessed by flow cytometry. Shaded histogram represents isotype-matched negative control Ab staining; open histogram represents a specific Ab staining. Results are representative of 4 experiments with different donors. TLR, Toll-like receptor.
stand TLR expression patterns in monocytes to study the effects of TLR agonists on DCs. Thus, our experiments were initiated to assess surface and intracellular TLR expressions of human monocytes. By flow cytometric analysis, we found that monocytes expressed surface TLR2 and TLR4, but not TLR3 and TLR8 (Fig 1, A). However, intracellular staining detected expression of not only TLR2 and TLR4, but also TLR3 and TLR8 (Fig 1, B), indicating TLR3 and TLR8 reside only intracellularly. There was no evidence of TLR7 or TLR9 expression (not shown). IFN-␥ and R848 upregulate TLR8 expression in monocyte-derived DCs. TLR8 signaling has been shown to synergize with IFN-␥ to activate DCs. To study the mechanism of its interaction with IFN-␥ or other possible TLR agonists, we examined regulation of expression of TLR8 messenger RNA (mRNA) in monocyte-derived DCs. We stimulated immature DCs with IFN-␥ or TLR agonists, respectively. On the basis of the human monocytes’ expression of TLR2, TLR3, TLR4, and TLR8, their corresponding ligand LTA, poly (I: C), LPS and R848 were used in the studies. DCs treated with IFN-␥ or R848 for 4 hours expressed large amounts (4- to 5-fold increase) of TLR8 mRNA, and LPS moderately upregulated TLR8 mRNA, whereas LTA and poly(I: C) had no effect on TLR8 expression (Fig 2). TLR agonists induce maturation and expression of DC costimulatory molecules. Because alteration in surface protein expression is an important step
in the maturation of DCs, we next investigated whether TLR agonists had an additive effect on expression of DC costimulatory molecules. Immature DCs were activated either with a single TLR agonist for 24 hours or primed first with a TLR agonist for 2 hours, then matured with LPS for an additional 20 to 24 hours. The surface phenotype was then analyzed by flow cytometry. DCs activated by a single TLR signal (Fig 3, A) displayed levels of costimulatory molecules comparable to those expressed on DCs induced by 2 TLR signals (Fig 3, B). All DCs showed similar low levels of CD14, and upregulated CD80, CD86, and CD83. This finding implies that single TLR signaling possesses the capacity to fully mature DCs, but contemporaneous signaling through additional TLR family members has little additional effect on expression of DC costimulatory molecules. TLR8 agonist R848 primes DCs for high level of IL-12 production. High levels of IL-12 secretion in human DCs require 2 signals,1 such as an initial priming signal supplied by IFN-␥ and a maturation signal provided by LPS or CD40L. The importance of IL-12 in tumor immunity prompted us to explore whether other signaling molecules could substitute for IFN-␥ in DC priming. Since TLR agonists have been shown to induce Th1 immunity, we therefore examined their capacity to prime DCs for IL-12 production. Immature DCs were primed with a single TLR agonist LTA, poly (I: C), LPS, or R848 separately for 2 hours and then matured with either LPS or CD40L for 16 to 20 hours. Culture
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Fig 2. TLR8 mRNA expression in human monocyte– derived immature DCs is regulated positively by IFN-␥ and R848. Overnight-cultured human monocytes (immature DCs) were treated with IFN-␥, LTA, poly (I: C), LPS, or R848, respectively, for 4 hours. Cells were harvested, and RNAs were extracted for analysis of expressions of TLR8 and GAPDH by Northern blots. Results are representative of 5 experiments. GAPDH, Glyceraldehyde-3-phosphate dehydrogenase; GM-CSF, granulocyte-macrophage colony-stimulating factor; IFN-␥, interferon gamma; LPS, lipopolysaccharide; LTA, lipoteichoic acid; TLR, toll-like receptor; poly (I: C), polyinosinic-polycytyidilyc acid.
Fig 3. DCs express comparable levels of costimulatory molecules when induced by either a single TLR signaling or multiple TLR agonists. Human monocytes were cultured in serum-free medium plus GM-CSF overnight (immature DCs); they were then treated with LTA, poly (I: C), LPS, or R848, respectively, for 24 hours (A); immature DCs were primed with LTA, poly (I: C), and R848 separately for 2 hours, then further matured with LPS for additional 20-24 hours (B). DCs were harvested and surface phenotype was analyzed by flow cytometry. For CD14, filled traces represent CD14 staining of monocyte controls (starting populations), and open traces represent CD14 staining of cultured DCs. For CD80, CD86 and CD83, filled traces represent isotype-matched negative control staining; open traces represent specific Ab staining. Results are representative of 5 experiments. GM/IL-4, granulocyte-macrophage colony-stimulating factor/ interleukin-4; IFN-␥, interferon gamma; IL-12, interleukin 12; LPS, lipopolysaccharide; LTA, lipoteichoic acid; poly (I: C), polyinosinic-polycytyidilyc acid; R848, resimiquod.
supernatants were harvested for measurements of IL-12p70 by ELISA. For comparison, we also treated immature DCs with TLR agonists alone for 24 hours or primed the DCs with IFN-␥ for 2 hours,
then matured the cells with TLR agonists, respectively, for 20 hours. Figure 4, A demonstrates that DCs induced by a single TLR agonist secreted no IL-12p70; DCs primed with IFN-␥ and then ma-
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Fig 4. TLR8 agonist R848 primes DCs for high level of IL-12 production. Immature DCs were treated with lipotechoic acid (LTA), poly (I: C), LPS, or R848 individually for 2 hours and then stimulated with either LPS (black bar) or CD40L (dotted bar) for 20 hours. For comparison, immature DCs were activated by LTA, poly (I: C), LPS, or R848 alone for 24 hours (white bar), or primed with IFN-␥ first for 2 hours and then stimulated with LTA, poly (I: C), LPS, or R848, respectively, for 20 hours (horizontal line bar). IL-12p70 secretions in culture supernatants were measured by ELISA (A). In separate experiments, cells were treated with a single TLR agonist for 4 hours or primed with TLR agonists individually for 2 hours and further activated by LPS for additional 2 hour. Total RNA was extracted for IL-12p35 and p40 expression analysis by Northern blot (B and C). Results are representative of 3 experiments. GAPDH, Glyceraldehyde-3-phosphate dehydrogenase; GM-CSF, granulocyte-macrophage colony-stimulating factor; IFN-␥, interferon gamma; IL-12, interleukin 12; LPS, lipopolysaccharide; LTA, lipoteichoic acid; poly (I: C), polyinosinic-polycytyidilyc acid; R848, resimiquod.
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tured with a TLR agonist produced high levels of IL-12p70 as expected. LTA, poly (I: C), and LPS, which trigger TLR2, TLR3, and TLR4, were unable to prime DCs for IL-12p70 production; only the TLR8 agonist R848 displayed the unique property of priming DC for high IL-12p70 production. In a parallel experiment we also examined the expression pattern of IL-12p35 and p40 mRNA. DCs activated by a single TLR agonist induced only IL12p40 mRNA transcript but no p35 (Fig 4, B); DCs primed with LTA or poly (I: C) and subsequently matured with LPS failed to induce IL-12p35 (Fig 4, C). In a separate experiment DCs primed with TLR7 agonist 7-Deaza-2’deoxyguanosine and matured with LPS also were unable to express IL12p35 mRNA (data not shown). In contrast, DCs primed with IFN-␥ or R848 followed by LPS maturation expressed both IL-12p35 and p40 genes (Fig 4, C). These findings suggest that induction of the IL-12 p35 gene requires 2 signals: Only TLR8 agonist R848 could replace IFN-␥ as a priming signal, synergizing with LPS or CD40L in the production of DC IL-12p70. R848 and LPS synergistically induce TNF-␣ and IL-6 secretion by DCs. We next tested whether other cytokines were induced by the TLR agonists. TNF-␣ and IL-6, 2 Th1 cytokines, also were induced synergistically by a combined treatment of R848 and LPS as shown in Figure 5. Interestingly unlike IFN-␥, R848 treatment of immature DCs stimulated significant IL-6 production (Fig 5). In addition, although poly (I: C) and LPS did not induce IL-12 production, there was evidence of production of both IL-6 and TNF-␣ (Fig 5). R848-primed DCs display Th1-polarizing capacity. To further study the functional ability of R848-primed DCs, we carried out allosensitization experiments to analyze their capacity to functionally polarize CD4⫹ T cells. Naïve allogeneic CD4⫹T cells were stimulated by CMM-induced DCs12 or DCs primed with R848, IFN-␥, or poly (I: C), respectively. Their functional phenotype was assessed by ELISA of culture supernatants. R848- or IFN-␥–primed DCs, which secreted high levels of IL-12, stimulated naïve CD4⫹T cells to produce large quantities of IFN-␥ with little IL-4, whereas poly(I: C) primed–DCs or CMM-DCs, which produced little or no IL-12, stimulated T cells to secrete much less IFN-␥, but abundant IL-4 (Fig 6,A). Thus, these 2 distinct types of DCs polarized CD4⫹ T cells into being characteristic of either the Th1 or Th2 phenotype. The Th1-polarized CD4⫹T cells induced by R848-primed DCs could be blocked by IL-12– neutralizing monoclonal antibody (Fig 6, B).
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Fig 5. R848 and LPS synergistically induce TNF-␣ and IL-6 secretion by DCs. Immature DCs were treated with IFN-␥, LTA, poly (I:C), LPS, or R848, respectively, for 24 hours (white bar), or they were primed with IFN-␥ LTA, poly (I: C), or R848 for 2 hours, followed by LPS treatment for 20 hours (black bar). Culture supernatants were collected for cytokine assays by ELISA. Results are representative of 5 experiments with different donors. IFN-␥, Interferon gamma; IL-12, interleukin 12; LPS, lipopolysaccharide; LTA, lipoteichoic acid; poly (I: C), polyinosinic-polycytyidilyc acid; R848, resimiquod; TNF-␣, tumor necrosis factor ␣.
These results demonstrate that IL-12–producing DCs primed by R848 have a high capacity to polarize CD4⫹ T cells toward a Th1 phenotype. R848-primed DCs confer CD8ⴙ T cells with an enhanced ability to recognize tumor cells. We have shown previously that IL-12–producing DCs generate high functional avidity CD8⫹ T cells, which have an enhanced ability to recognize tumor cells. We therefore tested whether R848-primed DCs also displayed this property. As Figure 7, A and C demonstrate, poly(I: C)–primed DCs or CMM-DCs, which produced little or no IL-12–induced CD8⫹ T cells, recognizing only T2 target cells pulsed with MART-1 (27-35), the peptide to which they were sensitized, but not tumor cells expressing MART-1. In contrast, IL-12–producing DCs primed by either R848 or IFN-␥ sensitized CD8⫹ T cells for large quantities of Ag-specific IFN-␥ release in response to both T2 cells pulsed with MART-1 (27-35) and melanoma cells positive for HLA-A2, which ex-
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Fig 6. R848-primed DCs display Th1-polarizing capacity. Naïve allogeneic CD4⫹ T cells were stimulated by CMMinduced DCs or DCs primed with R848, IFN-␥, or poly (I: C), respectively, and matured with CD40L. On day 6, activated T cells were restimulated with plate-bound anti-CD3 and anti-CD28 for 24 hours. A, Supernatants were analyzed for cytokine secretion by ELISA. B, ELISA analysis of supernatants from restimulated T cells cocultured with DCs generated by R848/CD40L in the presence of IL-12–neutralizing Ab or isotype-matched control mucosal antibody. Results are representative of 3 experiments. Ab, antibody; CMM-DC, cytokine maturation mixture-dendritic cell; IFN-␥, interferon gamma; IL-12, interleukin 12; LPS, lipopolysaccharide; LTA, lipoteichoic acid; poly (I: C), polyinosinic-polycytyidilyc acid; R848, resimiquod.
pressed endogenous melanoma antigen MART-1. IL-12–neutralizing antibody blocked such tumor recognition. However, these T cells still recognized target T2 cells pulsed with the relevant peptide (Fig 7, B and D). Again, these results demonstrate the central role of IL-12 in tumor recognition by CD8⫹ T cells. DISCUSSION Mature DCs secreting high levels of IL-12p70 have the capacity to sensitize CD8⫹ T cells to tumor-associated antigens and endow them with enhanced antigen sensitivity (ie, functional avidity).4 Indeed, we demonstrated recently that this increase in functional avidity of CD8⫹ T cells is critical for direct recognition and destruction of tumor cells. These recent findings regarding the importance of IL-12 p70 secretion by DCs and the subse-
quent induction of T cells with high functional avidity already is serving as the basis for an ongoing clinical vaccine trial against ductal carcinoma in situ (DCIS). Here, IL-12 p70 –secreting DCs activated with IFN-␥ and LPS are pulsed with HER-2/ neu peptides and then administered intranodally to subjects with this carcinoma. These DCs are sensitizing high functional avidity anti–HER-2/neu T cells in vivo that are inducing apparent clinical responses (manuscript in preparation). To date IFN-␥, ssRNA, or contact with natural killer cells have been the principal ways to prime DCs for high secretion of IL-12 p70 upon subsequent maturation.6,10 TLR agonists have been shown to mature DC, and some paired combinations of TLR agonists (but not others) can induce DCs to secrete large amounts of IL-12 p70 as well.16,17 We demonstrate here that the synthetic
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Fig 7. R848 primes DCs for high level of IL-12 production, accounting for enhanced tumor recognition by sensitized CD8⫹ T cells. Immature DCs were primed with IFN-␥, R-848, or poly (I: C), respectively, for 2 hours and further matured with CD40L for 20 hours. They were pulsed with peptide MART-1 (27-35) 2 hours before harvest. DCs were then cocultured with purified CD8⫹ T cells. After 7 days, T cells were harvested and tested for reactivity (IFN-␥ release) to target melanoma cell lines or to T2 cells pulsed with relevant (MART-1 [27-35]) and irrelevant peptides (A and C) by ELISA analysis of 24-hour culture supernatants. Adding neutralizing anti–IL-12 to the coculture of T cells and DCs blocked the tumor recognition of CD8⫹ T cells induced by R848-primed and CD40L-matured DCs. However, these T cells still recognized target T2 cells pulsed with optimal level of MART-1 (27-35) peptide (B and D). Results are representative of 4 experiments from different donors. Ab, antibody; CD40L, CD40 ligation; CMM-DC, cytokine maturation mixture-dendritic cell; IFN-␥, interferon gamma; IL-12, interleukin 12; poly (I: C), polyinosinic-polycytyidilyc acid; R848, resimiquod
TLR8 agonist R848 primes myeloid-derived DCs for high-level production of IL-12 upon subsequent maturation with LPS or CD40L. Our results agree with those of Gautier et al16 as well as Napolitani et al,17 demonstrating that multiple TLR agonists can stimulate IL-12 production by DCs. However, the mechanism by which TLRs prime DCs for high-level production of IL-12 is not entirely clear. It has been suggested that type-I interferons induced by TLRs provide priming signals in the DCs,15 but not all studies have observed evidence of this effect.16 In that regard, the TLR3 agonist poly (I: C) used in this study induces type-I interferon in these DCs,6 but in amounts insufficient to prime DCs for IL-12 production. We also made the interesting observation that both IFN-␥ and R848 increased dramatically the
levels of TLR8 expression in the DCs. Because each of these signals can cooperate, both with each other and individually in combination with other TLR agonists, to induce high-level secretion of IL12, possible biologic relationships between their respective signaling pathways are suggested. Indeed, preliminary data from our laboratory suggest that both IFN-␥ and R848 can greatly increase the expression of both interferon regulatory factors 1 and 8, 2 nuclear factors that are critical for the regulation of IL-12 expression.18 But what are the possible roles of TLR agonists in improving vaccines against malignancies and infectious agents? Natural ligands for TLRs often are complex biopolymers that may be difficult to purify in sufficient quantity from cultured organisms, relatively expensive to synthesize, and sometimes easily de-
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graded. For example, whereas our studies show particular promise for TLR8 agonists in priming DCs for IL-12 secretion and subsequent sensitization of high functional avidity, tumor-recognizing CD8⫹ T cells, the natural ligand, ssRNA, is broken down quickly by nucleases. Fortunately, small organic molecules such as R848 can make an excellent substitute, and other small organic pharmaceuticals that exploit the capacity to activate various TLRs undoubtedly are on the way. Identification of pathways leading to IL-12 production may provide additional insights into design and use of such new adjuvants for treating cancer and infectious diseases that require Th 1–polarized T cells. REFERENCES 1. Snijders A, Kalinski P, Hilkens CM, Kapsenberg ML. Highlevel IL-12 production by human dendritic cells requires two signals. Int Immunol 1998;10(11):1593-8. 2. Liu, J, Cao S, Herman LM, Ma X. Differential regulation of interleukin (IL)-12 p35 and p40 gene expression and interferon (IFN)-{gamma}-primed IL-12 production by IFN regulatory factor 1. J Exp Med 2003;198(8):1265-76. 3. Kalinski P, Vieira P, Schuitemaker JH, de Jong EC, Kapsenberg ML. Prostaglandin E2 is a selective inducer of interleukin-12 p40 (IL-12p40) production and an inhibitor of bioactive IL-12p70 heterodimer. Blood 2001;97(11):3466-9. 4. Xu S, Koski G, Faries M, et al. Rapid high efficiency sensitization of CD8⫹ T cells to tumor antigens by dendritic cells leads to enhanced functional avidity and direct tumor recognition through an IL-12-dependent mechanism. J Immunol 2003; 171(5):2251-61. 5. Dutoit V, Rubio-Godoy V, Dietrich PY, et al. Heterogeneous T-cell response to MAGE-A10254-262: high avidity-specific cytolytic T lymphocytes show superior antitumor activity. Cancer Res 2001;61(15):5850-6. 6. Koski GK, Kariko K, Xu S, Weissman D, Cohen PA, Czerniecki BJ. Cutting edge: innate immune system discriminates between RNA containing bacterial versus eukaryotic structural features that prime for high-level IL-12 secre-
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