Melanoma patients may exhibit a TH2-skewed cytokine profile within blood and tumor-infiltrating lymphocytes. Therapies that induce beneficial TH1-type tumor-specific immune responses, therefore, are highly desirable. Dendritic cells (DC) are widely used as immune adjuvants for cancer. Before their administration, DC are generally induced to mature with a cocktail of recombinant cytokines [interleukin (IL)-1β, tumor necrosis factor α, and IL-6] and prostaglandin E2 (PGE2), which is added to preserve the ability of DC to migrate to draining lymph nodes. However, PGE2 suppresses the production of IL-12p70, a cytokine essential for differentiation of TH1 responses. In this study, human DC were transfected with IL-12p70 mRNA and tested for their ability to alter the TH2 type bias manifested by blood T cells of patients with melanoma. Transfected DC secreted high levels of bioactive IL-12p70, as indicated by their capacity to enhance natural killer cell activity, skew TH1 responses in allogeneic mixed lymphocyte reactions through reduction of IL-4 and IL-5, and prime CD8+ T cells to the melanoma-associated antigen Melan A/MART-1. Furthermore, T-cell lines primed in vitro from the blood of melanoma patients showed strong type 2 skewing that was dramatically reversed by IL-12p70 transfection of autologous DC. Thus, IL-12p70 transfection of clinical DC preparations may enhance type 1 antitumor responses and may thereby contribute to effective immune-based therapy. [Cancer Res 2008;68(22):9441–50]

Substantial evidence exists to indicate that T-cell responses can mediate the suppression and elimination of tumors in animal models and in human subjects (13). Tumors in subjects with progressive cancer may evade these responses through various mechanisms, including antigenic variation and expression of immune-suppressive factors such as interleukin (IL)-10, vascular endothelial growth factor, transforming growth factor β, and programmed death ligand 1 (4). Additionally, the cytokine milieu in the tumor-bearing host can determine the particular type of immune response mounted and, thus, the efficiency of the antitumor response in eradicating tumor cells.

Certain tumors, including melanoma, may be associated with production of IL-5, IL-4, and IL-13 by CD4 and CD8 T cells (TH2 type immune responses), thereby adding an additional immunomodulating step that can repress host antitumor responsiveness (59). McCarter and colleagues (7) showed that conditioned medium from in vitro cultures of melanoma cells could drive T-cell lines toward a TH2 phenotype in coculture with monocyte-derived dendritic cells (MDDC). Notably, in a clinical trial in which human subjects with melanoma received autologous MDDC transfected with mRNA encoding tumor antigen, antigen-specific T cells produced significantly greater amounts of IL-5 and IL-13 after vaccination, whereas there was no significant increase in IFN-γ or tumor necrosis factor-α (TNF-α) production [type 1 (TH1) T-cell response] in the same patients (10). Thus, effective tumor vaccines should induce T-cell responses to a broad range of epitopes derived from essential oncogenic proteins while promoting proinflammatory functions to overcome tumor-associated immune deviation. A widely investigated method of therapeutic antitumor vaccination is dendritic cell (DC) immunotherapy (11). DC in their various forms are the most potent antigen-presenting cells for the induction of immune responses in animals and humans. DC may be loaded with tumor antigens by exposure to recombinant tumor proteins or tumor lysate or by DNA transfection, retroviral transduction, or mRNA transfection. In particular, transfection with mRNA is a promising method for the delivery of multiple tumor antigens and immunomodulatory molecules to DC in a safe and efficient manner (12).

The type of DC most widely used for immunotherapy is the monocyte-derived DC (MDDC), which is differentiated in vitro from peripheral blood monocyte precursors in the presence of IL-4 and granulocyte macrophage colony-stimulating factor (GM-CSF). Following differentiation, DC must undergo a maturation step before they gain the ability to induce and enhance antitumor T-cell responses (13, 14). For clinical production of MDDC, maturation is usually induced by a cytokine cocktail that includes recombinant TNF-α, IL-1β, and IL-6. Prostaglandin E2 (PGE2) is often added (15) because it preserves the migratory ability of maturing MDDC as shown in in vitro studies (16). However, PGE2 inhibits the production of bioactive IL-12p70 (1719). IL-12p70 is a heterodimeric cytokine that plays an essential role in innate and adaptive immune responses that mediate antitumor resistance (20). It functions in the regulation of the helper T-cell response to optimally induce, activate, and expand a TH1 response (21). IL-12p70 also enhances the CD8+ T-cell cytolytic response (22), and its antiangiogenic properties further contribute to an important role in antitumor responses (20, 23). Many experimental mouse models have illustrated the effectiveness of IL-12p70 in inhibiting tumor growth, improving the survival of tumor-bearing mice, and inducing effective tumor-specific immunity (20). Experiments in vitro and in vivo have opened the way for clinical trials using systemic recombinant human (rHu) IL-12p70 as adjuvant antitumor therapy (2426). The trials to date have been promising and have shown some evidence of effective immune responses [including enhancement of natural killer (NK) cell activity, T-cell proliferation, and IFN-γ production] and clinical regression of tumors with partial and complete responses (20, 24, 26). Systemic administration of IL-12p70, however, has inherent and considerable toxicities (27). Furthermore, lack of availability of clinical grade rHu IL-12p70 may pose a limitation to additional trials. Therefore, new approaches to improve the immunogenicity of PGE2-treated MDDC are urgently required.

In this study, we investigated the effect of transfecting human MDDC with a single synthetic mRNA encoding IL-12p70. We evaluated the ability of transfected MDDC to promote proinflammatory functions crucial for the elimination of tumors in vivo. These include NK cell activation, priming and expansion of tumor-specific T-cell responses, and type 1 skewing of T cells derived from melanoma patients. Our results suggest that IL-12p70 mRNA transfection of PGE2-treated MDDC promotes innate immunity and reverses tumor-associated immune evasion by enhancing the production of IFN-γ and simultaneously inhibiting the production of the type 2 cytokines IL-4, IL-5, and IL-13. Significantly, these effects are demonstrable at both the CD4+ and CD8+ T-cell levels.

Human subjects. Subjects with melanoma were recruited from the New York University Cancer Institute, and peripheral blood cell samples were acquired by leukapheresis. HLA-A*0201–positive stage IIB, IIC, and/or III melanoma patients that were clinically free of disease but at high risk for recurrence were included. Blood samples from healthy subjects were acquired by venipuncture or leukapheresis. Leukaphereses from normal healthy subjects were obtained from BRT Laboratories. Approval was obtained from the New York University School of Medicine Institutional Review Boards (IRB) for these studies. For NK cell studies, healthy donors were recruited at the Massachusetts General Hospital; the Massachusetts General Hospital IRB approved the study, and each subject gave informed consent for participation in the study. In all cases, informed consent was provided according to the Declaration of Helsinki.

Cell culture. Peripheral blood mononuclear cells (PBMC) were purified by Ficoll-Hypaque density gradient centrifugation, washed, and frozen in sterile aliquots in medium supplemented with 10% human albumin USP and 10% DMSO. All cells were grown in RPMI medium (Cellgro) supplemented with 1 mmol/L HEPES (Life Technologies, Inc.) and 20 μg/mL gentamicin (Life Technologies). RPMI with 1% pooled human plasma (Valley Biomedical) was used for culture of DC, and RPMI with 5% pooled human serum (Valley Biomedical) was used for all other cell assays. For B lymphoblastoid cell lines, 10% fetal bovine serum (Life Technologies) was used. T-cell lines were grown and assayed in 5% pooled human serum.

DC preparation. PBMC were obtained from buffy coats (New York Blood Center) or leukapheresis products (BRT labs-MD) by Ficoll-Paque Plus gradient centrifugation (Pharmacia). PBMC were plated in tissue culture plates (Falcon) at 35 × 106 per plate in RPMI 1640 supplemented with 20 μg/mL gentamicin, 10 mmol/L HEPES, and 1% human plasma. Cells were allowed to adhere for 2 h at 37°C and 5% CO2. Nonadherent cells were then removed by several washes. The monocyte-enriched fraction was supplemented with 116 IU/mL rHu GM-CSF (Immunex) and 15 to 300 IU/mL of rHu IL-4 (R&D) on days 2 and 4 of culture. Immature DC were harvested on day 5 of culture. DC were matured using a cocktail consisting of 5 ng/mL IL-1β, 5 ng/mL TNF-α, 150 ng/mL IL-6 (all from R&D), and 1 μg/mL PGE2 (Sigma), which was added directly to DC on day 5 of culture for 24 h.

Preparation of IL-12 construct. A single IL-12p70 open reading frame encoding the IL-12 p40 and p35 subunits joined by an elastin linker was subcloned by PCR from the pORF-hIL-12 vector (Invivogen) into the XhoI and PacI sites of transcriptional template vector psp73-Sph/A64 (28). PCR was done using primers 5′-TATATACTCGAGAGGAGGGCCACCATGGGT and 5′-GCGCGCTTAATTAACCATTAGGAAGCATTCAGATAGC.

Transfection of antigen-presenting cells. For transfection, antigen-presenting cells (APC) were washed thrice and resuspended to a concentration of 3.3 × 106/mL in OptiMEM medium (Invitrogen). APCs (106) were incubated with mRNA on ice for 10 min and then electroporated in a 0.2-cm cuvette with a GenePulser Xcell (Bio-Rad) at 400 V/0.75 ms, square wave protocol.

NK cell experiments. MDDC were prepared from the blood of normal donors and transfected with IL-12p70 mRNA or irrelevant control mRNA. Transfected MDDC were cocultured with autologous purified NK cells or autologous whole PBMC as described. Untouched NK cells were negatively isolated from PBMC with a RosetteSep NK kit (StemCell Technologies, Inc.). For the Elispot assay, NK cells and MDDC were cocultured at a ratio of 1:1 overnight. For the flow-based assay, MDDC were cocultured with autologous PBMC overnight in 5% human serum without addition of brefeldin A. PBMC were then stained for CD3 and CD56, fixed, and analyzed by fluorescence-activated cell sorting (FACS). To measure cytolytic activity, immature MDDC were transfected with IL-12p70 or control mRNA and rested at 37°C for 2 h. K562 and transfected MDDC targets were labeled with Na2(51CrO4) for 1 h at 37°C and incubated with autologous NK effectors at the indicated ratios for 4 h at 37°C. The percent lysis was calculated as [(sample count − spontaneous release) / (maximal release − spontaneous release)] × 100.

Isolation and quantification of mRNA from T-cell lines. Total RNA was extracted with the RNeasy mini kit (Qiagen) according to the manufacturer's protocols. Complementary DNA was generated using an oligo dT primer and Powerscript RT enzyme (BD Clontech) at 42°C for 60 min. Gene expression was quantified by real-time PCR on an Mx3005P thermocycler (Stratagene) using Full Velocity SYBR Green 2× mix (Stratagene) and gene-specific primers (Supplementary Table S1). Following denaturation (10 min, 95°C), DNA was amplified using 40 cycles at 95°C, 60°C, and 72°C (20 s each). Primer pairs were designed to span intron boundaries and were tested to verify specificity for mRNA products, and not genomic DNA, based on size and sequencing of each product. All primers were purchased from Invitrogen. Results were normalized to hypoxanthine phosphoribosyltransferase using a 2ΔΔCt method and are displayed as relative expression values in arbitrary units.

Intracellular cytokine staining. T-cell lines generated from priming cultures were stimulated with 2 μmol/L of MART-1(21–35) peptide (YTTAEEAAGIGILTV). After 1 h, 10 μg/mL brefeldin A (Sigma) was added to the stimulated cells. Five hours later, cells were stained for surface markers (CD3, CD4, and CD8; Becton Dickinson), then fixed with 4% paraformaldehyde, permeabilized with 0.1% saponin, and stained for intracellular cytokines. Cells were acquired with FACSCalibur and LSRII and analyzed using CellQuest and FlowJo.

Allogeneic mixed lymphocyte reactions. Immature or cytokine cocktail–matured DC were either mock electroporated or electroporated with 10 μg IL-12p70 mRNA per 106 cells (as above), and then mixed in triplicate with allogeneic CD4+ naïve T cells derived by magnetic depletion of CD19, CD14, CD56, and CD45RO (Miltenyi Biotech). rHu IL-12p70 (R&D Systems) was added to cultures on day 0. After 5 to 7 d at 37°C, after rigorous washing, CD3/CD28 beads (Dynal, Invitrogen) were added to the cultures for 14 to 24 h. Supernatants from stimulated cells were harvested and analyzed for cytokines secreted using the TH1/TH2 cytokine bead array (BD).

Priming experiments. DC were grown from melanoma patients' PBMC as described above. Cytokine cocktail–matured DC were harvested and transfected with 15 μg LysoMART-1 mRNA or with 15 μg LysoMART-1 mRNA plus 10 μg IL-12p70 mRNA. The DC were then irradiated at 35 Gy. DC were cocultured with autologous nonadherent PBMC fractions after magnetic depletion of CD25+ cells (Miltenyi Biotech) in the presence of IL-6 (1,000 units/mL; R&D Systems). Extra DC were frozen and used for restimulation. The cultures were restimulated every 7 to 10 d with 10 units/mL IL-2 and 5 ng/mL IL-7 (final concentration) using the same DC conditions and tested by intracellular cytokine staining or cytokine bead array for peptide-specific cells, or were stimulated with phorbol 12-myristate 13-acetate (PMA) plus ionomycin or CD3/CD28 beads (Dynal) for cytokine secretion assays.

Cytokine bead array. Stimulated T cells were incubated at ∼106/mL overnight, and samples of supernatants were acquired and frozen for future analysis. Thawed samples were tested with the human TH1/TH2 cytokine bead array (BD PharMingen) and used to measure IL-4, IL-5, IFNγ, and TNF-α after T-cell stimulations.

IL-12p70 measurement by ELISA. Cytokine cocktail–matured DC were electroporated with 10 μg of IL-12p70 or GFP mRNA as negative control. DC were resuspended in RPMI with 1% human plasma (1% plasma) at 106 cells/mL and cultured at 37°C, 5% CO2 with 95% humidity. At the indicated time points, supernatants were harvested and replaced with fresh, warm 1% plasma. The cumulative concentration of IL-12p70 was determined by culturing DC separately for various lengths of time before harvesting supernatants for ELISA (IL-12p70 ELISA kit, BD).

LysoMART-1 mRNA production. A transcriptional template for chimeric lysosome-targeted Melan-A/MART-1 (LysoMART-1) was constructed as previously described for other antigens (29, 30). We used a vector (pLyso) containing the following sequential elements: a T7 polymerase promoter, a Kozak ribosome binding motif including the start codon of the open reading frame, the endoplasmic reticulum–targeting translocation signal of heat shock protein gp96, an antigen insertion locus, the lysosomal targeting motif of lysosome-associated membrane protein 1, a stop codon, and a polyadenylate tail sequence. Synthetic mRNA was prepared by transcription from a linear DNA template with the Message Machine kit (Ambion).

Deriving tumor-infiltrating lymphocytes. To isolate melanoma tumor-infiltrating lymphocytes (TIL), fresh tumor tissue was minced under sterile conditions and plated in R10 medium (RMPI 1640 supplemented with 10% heat-inactivated human AB serum) at 2 mL/well in wells of a well plate, adding one or two 1-mm cubes of tissue per well. Wells were supplemented with IL-2 (Roche) to a final concentration of 150 units/mL to grow TIL. Cells were then cultured at 37°C, 5% CO2, replenishing IL-2 twice per week, and wells were split when necessary. TIL were harvested once 24 confluent wells were obtained and were then counted and frozen in aliquots in R10-pooled human serum/10% DMSO supplemented with 150 units/mL IL-2 and stored in a liquid nitrogen freezer.

MDDC secrete IL-12p70 on transfection with IL-12p70 mRNA. Previous reports have shown that transfection with mRNA is an efficient way to modify MDDC to express antigens or costimulatory molecules (31). We constructed a template vector encoding a single open reading frame representing the p35 and p40 subunits of IL-12p70 joined together by an elastin linker under the control of the T7 polymerase promoter. We used this template to synthesize mRNA in vitro and transfect MDDC by electroporation. As shown in Fig. 1, MDDC secreted significant levels of IL-12p70 on transfection with IL-12p70 mRNA, but not GFP mRNA. The majority of the secretion occurred within 6 hours after transfection. Additionally, MDDC frozen following transfection with IL-12p70 retained IL-12 secretory ability after thawing (Fig. 1C). Consistent with previous reports (24, 27), we found that mRNA capped with anti-reverse cap analogue (ARCA) induced prolonged secretion compared with conventional cap analogues (data not shown).

Figure 1.

IL-12p70 mRNA–transfected MDDC secrete high amounts of IL-12. Cytokine cocktail–matured DC were electroporated with 10 μg IL-12p70 or control GFP mRNA and cultured at a density of 106 cells/mL. Concentrations of IL-12p70 were measured by ELISA. A, rate of IL-12 secretion was determined by collecting supernatants from cultures at 6, 24, and 36 h after electroporation and replacing with fresh, warm medium. Rate (nanograms per hour) was calculated by dividing the amount of IL-12p70 produced per 106 DC by the number of hours in the time interval. Symbols represent distinct transfected APC populations (DC and B lymphoblastoid cell line). B, cumulative concentration of IL-12 was determined by culturing several preparations of DC or B lymphoblastoid cell line for 36 h and collecting supernatants at the end of the culture period. C, IL-12p70 mRNA–transfected MDDC retain IL-12 secretory ability on freeze-thawing. MDDC were transfected with IL-12p70 and frozen after 1 h of culture at 37°C. Frozen MDDC were subsequently thawed and IL-12p70 concentrations measured following thawing. EP, electroporation. Representative of four separate experiments.

Figure 1.

IL-12p70 mRNA–transfected MDDC secrete high amounts of IL-12. Cytokine cocktail–matured DC were electroporated with 10 μg IL-12p70 or control GFP mRNA and cultured at a density of 106 cells/mL. Concentrations of IL-12p70 were measured by ELISA. A, rate of IL-12 secretion was determined by collecting supernatants from cultures at 6, 24, and 36 h after electroporation and replacing with fresh, warm medium. Rate (nanograms per hour) was calculated by dividing the amount of IL-12p70 produced per 106 DC by the number of hours in the time interval. Symbols represent distinct transfected APC populations (DC and B lymphoblastoid cell line). B, cumulative concentration of IL-12 was determined by culturing several preparations of DC or B lymphoblastoid cell line for 36 h and collecting supernatants at the end of the culture period. C, IL-12p70 mRNA–transfected MDDC retain IL-12 secretory ability on freeze-thawing. MDDC were transfected with IL-12p70 and frozen after 1 h of culture at 37°C. Frozen MDDC were subsequently thawed and IL-12p70 concentrations measured following thawing. EP, electroporation. Representative of four separate experiments.

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Transfection of MDDC with IL-12p70 mRNA enhances NK cell response to class I–negative targets. To determine the bioactivity of the secreted IL-12p70 itself, we tested whether immature DC transfected with IL-12p70 were able to enhance NK cell activation. NK cells were isolated by negative selection from PBMC, and transfected MDDC were mixed 1:1 with NK cells in the presence or absence of K562 target cells. As shown in Fig. 2A, IL-12p70–transfected DC enhanced K562-induced IFN-γ secretion by autologous NK cells to a significantly greater extent than mock-transfected DC.

Figure 2.

IL-12p70 produced by transfected MDDC is bioactive and enhances target-specific responses of NK cells. A, NK cells isolated from PBMC of a normal donor were incubated with autologous mock-transfected or IL-12p70–transfected MDDC in the presence or absence of class I–negative K562 targets. rHu IL-12 was added to cocultures of NK cells and mock-transfected MDDC as an additional control. IFN-γ production, a measure of NK cell activation, was subsequently measured in an overnight ELISpot assay. SFC, spot-forming cells. B, PBMC from a normal donor were cocultured overnight with autologous IL-12p70–transfected MDDC in the presence or absence of K562 targets as shown. The degree of NK cell activation was determined by measuring CD69 expression on CD3CD56+ lymphocytes with FACS. The percentage of CD69+ cells is shown within the gate. C, mean fluorescence intensity of CD69 on CD3CD56+ NK cells and CD3+CD56 T cells. Enhancement of activation by IL-12p70 was specific for CD3CD56+ NK cells, as there was no change in the activation state of CD3+CD56 T cells. Results are representative of three similar experiments. D, IL-12 production by immature MDDC increases the cytolytic activity of autologous NK cells. Immature MDDC were transfected with IL-12p70 or control mRNA, labeled with 51Cr, and incubated with fresh autologous NK cells for 4 h in a chromium release assay; untransfected K562 targets were used as a positive control for cytolytic activity. The percent lysis was calculated as [(sample count − spontaneous release) / (maximal release-spontaneous release)] × 100.

Figure 2.

IL-12p70 produced by transfected MDDC is bioactive and enhances target-specific responses of NK cells. A, NK cells isolated from PBMC of a normal donor were incubated with autologous mock-transfected or IL-12p70–transfected MDDC in the presence or absence of class I–negative K562 targets. rHu IL-12 was added to cocultures of NK cells and mock-transfected MDDC as an additional control. IFN-γ production, a measure of NK cell activation, was subsequently measured in an overnight ELISpot assay. SFC, spot-forming cells. B, PBMC from a normal donor were cocultured overnight with autologous IL-12p70–transfected MDDC in the presence or absence of K562 targets as shown. The degree of NK cell activation was determined by measuring CD69 expression on CD3CD56+ lymphocytes with FACS. The percentage of CD69+ cells is shown within the gate. C, mean fluorescence intensity of CD69 on CD3CD56+ NK cells and CD3+CD56 T cells. Enhancement of activation by IL-12p70 was specific for CD3CD56+ NK cells, as there was no change in the activation state of CD3+CD56 T cells. Results are representative of three similar experiments. D, IL-12 production by immature MDDC increases the cytolytic activity of autologous NK cells. Immature MDDC were transfected with IL-12p70 or control mRNA, labeled with 51Cr, and incubated with fresh autologous NK cells for 4 h in a chromium release assay; untransfected K562 targets were used as a positive control for cytolytic activity. The percent lysis was calculated as [(sample count − spontaneous release) / (maximal release-spontaneous release)] × 100.

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As an alternative way to measure NK cell activation, we cocultured transfected immature MDDC from a normal donor with autologous PBMC in the presence or absence of K562 targets. After 16 hours of coculture, activation of NK cells was determined by FACS staining for CD69, an activation antigen. As shown in Fig. 2B, IL-12p70–transfected MDDC induced a 2- to 3-fold increase in CD69hi NK cells compared with mock-transfected MDDC. The increased activation was specific to NK cells, as the mean fluorescence intensity of CD69 staining specifically increased on NK cells (CD56+CD3) but not on T cells (CD56CD3+; Fig. 2C). Furthermore, transfection of immature MDDC with IL-12p70 produced a modest enhancement in the cytolytic activity of autologous NK effectors (Fig. 2D).

Transfection of MDDC with IL-12p70 mRNA enhances priming and expansion of antigen-specific T cells. An important function of IL-12p70 is to skew primary immune responses toward the TH1 type and away from the TH2 type. Therefore, we tested whether IL-12p70 transfection of immature MDDC could skew the priming of naïve T cells in vitro. We first used immature DC, which by themselves do not efficiently induce TH1 responses. Naïve (CD45RO) CD4+ T cells were cocultured with allogeneic MDDC previously transfected with IL-12p70 or control mRNA. In some cocultures, recombinant IL-12p70 was added, as shown in Fig. 3. After 5 to 7 days, T cells were stimulated with anti-CD3 antibody, and the production of representative TH1 (TNF-α and IFN-γ) and TH2 (IL-4, IL-5) cytokines was measured using a cytokine bead array. In contrast to control DC alone, IL-12p70 produced by transfected DC or added as recombinant protein inhibited production of the TH2 cytokine IL-5 (Fig. 3A). IFN-γ production was unchanged or increased in the presence of transfected or exogenous IL-12, respectively, indicating that the decrease in IL-5 production was specific and not a result of T-cell death secondary to the effects of IL-12 (Fig. 3B,ii). The experiments were repeated using cytokine cocktail–matured MDDC. Similar results were obtained confirming that IL-12 enhances TH1 skewing by inhibiting the production of the TH2 type cytokine IL-5 while enhancing TH1 cytokine expression. To confirm the importance of IL-12 in skewing the T-cell response away from TH2, MDDC were matured by the toll-like receptor agonists poly(inosinic-cytidylic) acid (poly I:C) and lipopolysaccharide (LPS), cytokine cocktail, or left immature. IL-12 secretion was measured by these various MDDC. Only poly I:C–matured MDDC produced IL-12p70 and were able to inhibit IL-5 secretion of T cells in an allogeneic mixed lymphocyte reaction (Fig. 3C).

Figure 3.

IL-12p70 transfection and recombinant IL-12p70 both inhibit IL-5 production in a primary allogeneic response. Immature MDDC were transfected with IL-12p70 or control GFP mRNA and cocultured with naïve allogeneic CD4+ T cells in the absence or presence of recombinant IL-12 for 7 d. At the end of the culture period, cells were stimulated with CD3/CD28 beads for 14 to 24 h, and cytokine production was determined by intracytoplasmic cytokine staining. A, i, IL-5 production by T cells induced in the presence or absence of IL-12p70 as indicated. ii, percent inhibition of IL-5 expression by IL-12p70–transfected DC or by addition of 1 ng/mL rHu IL-12. T-cell/DC ratio was 30:1. Inhibition was calculated as a percentage: [(IL-5)no IL-12 condition − (IL-5)with IL-12] / (IL-5)no IL-12 condition. Inhibition was significant for both recombinant and transfected IL-12 (P = 0.014 and P = 0.005, respectively). B, i, in contrast to IL-5, TNF-α production by allogeneic T cells is not inhibited by IL-12p70 transfection of MDDC. Results are representative of three experiments. ii, IFN-γ was measured from the primed T cells elicited in A. IFN-γ was measured using cytokine bead array assay (BD). Results are representative of three separate experiments. MFI, mean fluorescence intensity. C, differentially matured MDDC were incubated with allogeneic CD4+CD45RA+ T cells at a ratio of 1:90 for 12 d. Subsequently, T cells were stimulated with anti–CD3/CD28 beads overnight, and the presence of IL-5 was detected by cytokine bead array assay. MDDC were left immature or matured with LPS, poly I:C, or mimic. Differentially treated MDDC secreted varying amounts of IL-12p70. Immature and mimic-matured MDDC failed to secrete IL-12p70 whereas LPS matured MDDC secreted 0 to 100 pg/mL of IL-12. Poly I:C induced IL-12p70 in the amounts of 250 pg/mL to 20 ng/mL (data from more than five experiments). D, PBMC from healthy donors were used to obtain MDDC. DC were matured using cytokine cocktail and used to prime autologous CD25+-depleted T cells at a T cell/DC ratio of 10:1. DC were transfected with LysoMART-1 mRNA or with LysoMART-1 mRNA plus IL-12p70 mRNA. T-cell cultures were restimulated twice and tested for specificity using LysoMART-1 transfected autologous DC. Supernatants of cocultures were evaluated for cytokine secretion. T cells primed in the presence of IL-12p70 secreted more IFN-γ and less IL-5. Results illustrated are proportions of concentrations of cytokine secreted based on the proportion of absolute values.

Figure 3.

IL-12p70 transfection and recombinant IL-12p70 both inhibit IL-5 production in a primary allogeneic response. Immature MDDC were transfected with IL-12p70 or control GFP mRNA and cocultured with naïve allogeneic CD4+ T cells in the absence or presence of recombinant IL-12 for 7 d. At the end of the culture period, cells were stimulated with CD3/CD28 beads for 14 to 24 h, and cytokine production was determined by intracytoplasmic cytokine staining. A, i, IL-5 production by T cells induced in the presence or absence of IL-12p70 as indicated. ii, percent inhibition of IL-5 expression by IL-12p70–transfected DC or by addition of 1 ng/mL rHu IL-12. T-cell/DC ratio was 30:1. Inhibition was calculated as a percentage: [(IL-5)no IL-12 condition − (IL-5)with IL-12] / (IL-5)no IL-12 condition. Inhibition was significant for both recombinant and transfected IL-12 (P = 0.014 and P = 0.005, respectively). B, i, in contrast to IL-5, TNF-α production by allogeneic T cells is not inhibited by IL-12p70 transfection of MDDC. Results are representative of three experiments. ii, IFN-γ was measured from the primed T cells elicited in A. IFN-γ was measured using cytokine bead array assay (BD). Results are representative of three separate experiments. MFI, mean fluorescence intensity. C, differentially matured MDDC were incubated with allogeneic CD4+CD45RA+ T cells at a ratio of 1:90 for 12 d. Subsequently, T cells were stimulated with anti–CD3/CD28 beads overnight, and the presence of IL-5 was detected by cytokine bead array assay. MDDC were left immature or matured with LPS, poly I:C, or mimic. Differentially treated MDDC secreted varying amounts of IL-12p70. Immature and mimic-matured MDDC failed to secrete IL-12p70 whereas LPS matured MDDC secreted 0 to 100 pg/mL of IL-12. Poly I:C induced IL-12p70 in the amounts of 250 pg/mL to 20 ng/mL (data from more than five experiments). D, PBMC from healthy donors were used to obtain MDDC. DC were matured using cytokine cocktail and used to prime autologous CD25+-depleted T cells at a T cell/DC ratio of 10:1. DC were transfected with LysoMART-1 mRNA or with LysoMART-1 mRNA plus IL-12p70 mRNA. T-cell cultures were restimulated twice and tested for specificity using LysoMART-1 transfected autologous DC. Supernatants of cocultures were evaluated for cytokine secretion. T cells primed in the presence of IL-12p70 secreted more IFN-γ and less IL-5. Results illustrated are proportions of concentrations of cytokine secreted based on the proportion of absolute values.

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We next evaluated the effect of IL-12p70 transfection on the induction of tumor antigen-specific T-cell responses by DC. The melanoma-associated antigen MART-1 was used as a model antigen. To enhance antigen presentation to CD4+ T cells, we expressed MART-1 as a chimeric fusion construct (LysoMART-1) with the lysosomal targeting sequence of lysosome-associated membrane protein 1, as described for other antigens (26). MDDC prepared from normal donors were transfected with LysoMART-1 mRNA only or with LysoMART-1 plus IL-12p70 mRNA and cocultured with autologous T cells for two cycles of stimulation at 1-week intervals. At the end of this period, the resulting T-cell lines were stimulated with autologous LysoMART-1–transfected MDDC, and cytokine production was analyzed by cytokine bead array. Priming with IL-12p70–transfected MDDC led to a significant increase in IFNγ production. A mean enhancement of 54-fold in the IFN-γ/IL-5 ratio was observed compared with control MDDC, which did not produce IL-12 (Fig. 3D).

MDDC derived from melanoma patients prime and expand IFN-γ–producing cells following transfection with mRNA encoding IL-12p70. TH2-skewed lymphocytes have been documented in circulating blood cells and TIL of melanoma patients (5, 8, 9). We grew TIL from resected melanoma patients and chose to focus our studies on one TIL cell line. TIL obtained by isolating and culturing T cells from a resected melanoma tumor in the presence of IL-2 had properties consistent with previous observations. The line contained both CD4+ and CD8+ T cells and produced large amounts of IL-4, but not IFN-γ, in response to PMA/ionomycin stimulation (Fig. 4A). Quantitative reverse transcription-PCR (RT-PCR) analysis to examine expression and/or production of CD40L, CD69, IFN-γ, IL-2, IL-5, IL-4, and IL-13 was also undertaken. Following stimulation, mRNA for IL-2, IL-5, and IL-13 was significantly up-regulated (Fig. 4B). The overall levels of these cytokines are also shown in Supplementary Table S2. IL-4 expression was constitutively high and no detectable up-regulation was detected following stimulation of the cells (data not shown). These results indicate that the line exhibited a TH2-like phenotype, inclusive of both CD4+ (TH2) and CD8+ (TC2) T cells.

Figure 4.

TIL isolated from a patient with metastatic melanoma secrete predominantly TH2-type cytokines. A, TIL were isolated from a resected melanoma, expanded in the presence of IL-2, and frozen for storage in liquid nitrogen. Thawed TIL were rested for 24 h in the absence of any cytokines. On stimulation with PMA plus ionomycin for 6 h, both CD8+ and CD4+ T cells secreted high levels of the IL-4 and little or no IFN-γ. T cells were tested by intracellular cytokine staining. Results are representative of three separate experiments. B, TIL were stimulated as above with PMA plus ionomycin and lysed. mRNA was extracted and subjected to RT-PCR. cDNA for 49 different T-cell products was measured by quantitative PCR, and values were normalized to the quantity of actin mRNA. Values shown represent those factors for which PMA treatment resulted in >5× induction of specific message.

Figure 4.

TIL isolated from a patient with metastatic melanoma secrete predominantly TH2-type cytokines. A, TIL were isolated from a resected melanoma, expanded in the presence of IL-2, and frozen for storage in liquid nitrogen. Thawed TIL were rested for 24 h in the absence of any cytokines. On stimulation with PMA plus ionomycin for 6 h, both CD8+ and CD4+ T cells secreted high levels of the IL-4 and little or no IFN-γ. T cells were tested by intracellular cytokine staining. Results are representative of three separate experiments. B, TIL were stimulated as above with PMA plus ionomycin and lysed. mRNA was extracted and subjected to RT-PCR. cDNA for 49 different T-cell products was measured by quantitative PCR, and values were normalized to the quantity of actin mRNA. Values shown represent those factors for which PMA treatment resulted in >5× induction of specific message.

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We next tested whether circulating blood T cells of melanoma patients are similarly skewed toward a TH2-like phenotype. We cocultured CD25+ depleted T cells isolated from the blood of four melanoma patients with autologous MDDC transfected with LysoMART-1-1 alone or with LysoMART-1 plus IL-12p70 mRNA. The T cells were stimulated every 7 days with MDDC identical to those used in the induction step. One week after the induction step or the first restimulation, T-cell responses were tested for specificity by cytokine bead array assay using a pool of overlapping peptides spanning the entire MART-1 antigen sequence. Transfection of MDDC with IL-12p70 mRNA skewed the phenotype of the resulting T-cell lines by enhancing production of IFN-γ and inhibiting production of IL-5 and IL-4 (data not shown). The resulting MART-1–primed T-cell lines were restimulated for an additional 7 days with autologous LysoMART-1 mRNA– or LysoMART-1 + IL-12p70 mRNA–transfected MDDC. They were evaluated for TH1/TH2 type skewing after nonspecific, polyclonal stimulation with PMA/ionomycin, and results for one representative patient are shown in (Fig. 5). Priming by IL-12p70–transfected MDDC strongly skewed T cells toward a TH1 phenotype, as indicated by a 50-fold enhancement in the ratio of IFN-γ/IL-13–producing CD8+ T cells (Fig. 5A) and CD4+ T cells (Fig. 5C). Notably, in the absence of IL-12p70, the majority of primed IL-2+ CD8+ and CD4+ T cells did not produce IFN-γ, whereas IL-12p70 transfection caused most IL-2+ CD8+ and CD4+ T cells to acquire the ability to produce IFN-γ (Fig. 5B and D, compare Bi with Biii and Di with Diii). In the absence of IL-12p70, the majority of IL-2+IFN-γ T cells instead produced IL-13 and IL-5, whereas IL-12p70 strongly curtailed IL-5 production in these cells (Fig. 5B i–iv and Di–iv). Interestingly, the amount of IL-5 secreted by T cells primed in the absence of IL-12 was increased after each round of DC stimulation (data not shown). Of note, the IFN-γ–producing cells were both CD8+ and CD4+ T cells, although the vast majority of antigen-specific T cells were found to be CD8+.

Figure 5.

IL-12p70–transfected DC preferentially induce Tc1 responses from PBMC of melanoma patients. MDDC from a melanoma patient were transfected with LysoMART-1 mRNA plus IL-12p70 mRNA (left column) or LysoMART-1 mRNA only (right column) and used to prime autologous T cells. T cells were restimulated once or twice with MDDC at 7- to 10-d intervals. Thirty days after induction, T-cell lines were stimulated by PMA/ionomycin, and cytokine production was determined by intracellular cytokine staining. Results shown are gated on CD8+ T cells (A and Bi–iv) or CD4+ T cells (C and Di–iv). A and C, IL-12p70 transfection enhanced IFN-γ and suppressed IL-13 production by induced T cells. B and D, IL-12p70 transfection reduced the frequency of IL-2+IFN cells and enhanced the frequency of IL-2+IFN-γ+ cells. For Bi-iv and Di-iv, cells within the top right and bottom right quadrants in B and D, respectively, were further analyzed for production of IL-5 and IL-13. Bi, iii and Di, iii, a minority of IL-2+IFN-γ+ cells from B and D produced IL-5. Bii, iv and Dii, iv, IL-2+IFN-γ cells shown in B and D produce TH2 type cytokines (IL-13 and IL-5), and IL-5 production by these cells was strongly suppressed by IL-12p70 transfection. Results are representative of four patients tested.

Figure 5.

IL-12p70–transfected DC preferentially induce Tc1 responses from PBMC of melanoma patients. MDDC from a melanoma patient were transfected with LysoMART-1 mRNA plus IL-12p70 mRNA (left column) or LysoMART-1 mRNA only (right column) and used to prime autologous T cells. T cells were restimulated once or twice with MDDC at 7- to 10-d intervals. Thirty days after induction, T-cell lines were stimulated by PMA/ionomycin, and cytokine production was determined by intracellular cytokine staining. Results shown are gated on CD8+ T cells (A and Bi–iv) or CD4+ T cells (C and Di–iv). A and C, IL-12p70 transfection enhanced IFN-γ and suppressed IL-13 production by induced T cells. B and D, IL-12p70 transfection reduced the frequency of IL-2+IFN cells and enhanced the frequency of IL-2+IFN-γ+ cells. For Bi-iv and Di-iv, cells within the top right and bottom right quadrants in B and D, respectively, were further analyzed for production of IL-5 and IL-13. Bi, iii and Di, iii, a minority of IL-2+IFN-γ+ cells from B and D produced IL-5. Bii, iv and Dii, iv, IL-2+IFN-γ cells shown in B and D produce TH2 type cytokines (IL-13 and IL-5), and IL-5 production by these cells was strongly suppressed by IL-12p70 transfection. Results are representative of four patients tested.

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Finally, the tumor antigen-specific responses in these induced T-cell lines were also measured. The MART-1–specific responses for two of the patients are shown in Fig. 6. Induction with IL-12p70–transfected MDDC produced an enhancement of 5- to 30-fold in the frequency of CD8+ T cells that produced IFN-γ in response to the immunodominant MART-1 peptide (Fig. 6A). IL-12p70 also skewed responses to MART-1 peptide toward TH1-type cytokine production as measured by cytokine bead array, specifically by enhancing production of IFN-γ and reducing production of IL-5 (Fig. 6B).

Figure 6.

IL-12–transfected DC enhance priming of MART-1–specific TH1 like cells. A, MDDC from HLA-A*0201 melanoma patients were transfected with LysoMART-1 mRNA only or with LysoMART-1 plus IL-12p70 mRNA and used to prime autologous T cells. T-cell lines from two patients shown were stimulated with 2 μmol/L of the HLA-A*0201–restricted MART-1(21–35) peptide in the presence of brefeldin A for 6 h, and IFN-γ production was determined by intracellular cytokine staining. B, culture supernatants of T-cell lines were evaluated for cytokine secretion following overnight stimulation in the absence or presence of 2 μmol/L MART-1(21–35) peptide.

Figure 6.

IL-12–transfected DC enhance priming of MART-1–specific TH1 like cells. A, MDDC from HLA-A*0201 melanoma patients were transfected with LysoMART-1 mRNA only or with LysoMART-1 plus IL-12p70 mRNA and used to prime autologous T cells. T-cell lines from two patients shown were stimulated with 2 μmol/L of the HLA-A*0201–restricted MART-1(21–35) peptide in the presence of brefeldin A for 6 h, and IFN-γ production was determined by intracellular cytokine staining. B, culture supernatants of T-cell lines were evaluated for cytokine secretion following overnight stimulation in the absence or presence of 2 μmol/L MART-1(21–35) peptide.

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In summary, the expression of IL-12p70 by cytokine cocktail–matured DC shifts the underlying type 2 balance in melanoma patients' T cells toward type 1. The ability of IL-12 to skew CD8+ and CD4+ T-cell responses by enhancing IFN-γ secretion, reducing the production of IL-5 and IL-13, and priming tumor-antigen specific T cells emphasizes the potential advantage of IL-12 mRNA–transfected DC as adjuvants in tumor immunotherapy.

Therapeutic induction of effective antitumor T-cell responses in human cancer patients will require one to overcome several hurdles. These include a relative paucity of tumor-specific antigens (compared with vaccination against infectious diseases), tumor antigen loss variants, and tumor-associated immune deviation and dysfunction. Various lines of evidence suggest that in vivo skewing of T-cell responses toward a TH2 type is an important mechanism of immune evasion in cancer patients (5, 6, 8, 9, 32, 33) and animal models (34). This skewing may limit the efficacy of immunotherapeutic approaches. Several reports indicate that, compared with TH1 skewing, TH2 skewing of the vaccine-induced tumor-specific response correlates with worse disease outcome (6, 9, 34). Along those lines, we found that a TIL line isolated from resected melanoma exhibits a strong TH2 phenotype, producing relatively large amounts of IL-4 and IL-13 compared with IFN-γ or TNF-α (Fig. 4). Taken together, these results suggest that both spontaneous and vaccine-induced tumor-specific T-cell responses in human subjects are often deviated toward a nonprotective type 2 phenotype. Ensuring induction of a strong type 1 response may be critical to the development of effective cancer vaccines.

The events that lead to induction of type 1 or type 2 immune responses are complex and multifactorial. One of the most important determinants of type 1 skewing is production of IL-12p70 during the induction phase of the response (35). However, the production of clinical grade DC that are competent to produce IL-12p70 has proved to be extremely difficult because MDDC matured in vitro lose the ability to migrate to the lymph node on clinical infusion unless they are matured in the presence of PGE2 (15, 36). PGE2, however, blocks IL-12p70 expression by MDDC (17). Thus, most MDDC presently used in clinical trials do not make IL-12p70, and this defect may impair the efficacy of such vaccines.

Several promising methods exist to load antigen-presenting cells with tumor-derived antigens to maximize the breadth of antigen-specific responses. In particular, transfection of MDDC with synthetic mRNA encoding tumor-derived antigens offers several advantages. Transfected MDDC present endogenously processed antigenic peptides. Second, mRNA transfection is far more efficient than DNA transfection of MDDC, with nearly 100% of MDDC expressing antigen of interest (29). Third, mRNA lacks the theoretical risks associated with chromosomal integration as occurs in DNA transfection or retroviral transduction. Finally, RNA transfection offers a relatively straightforward method to alter expression of immunomodulatory factors to enhance DC function. Thus, mRNA transfection has been used to produce expression of proinflammatory factors [including IL-12 (37), OX-40L (38), and constitutively active toll-like receptor 4 (39, 40)] and small interfering RNA has been used to prevent expression of inhibitory factors (e.g., suppressor of cytokine signaling; ref. 41). RNA transfection may present a general tool to derive DC capable of overcoming the various challenges associated with antitumor vaccination. Recently Bontkes and colleagues (37) showed that transfecting MDDC with IL-12p70 mRNA could cause MDDC to produce clinically relevant amounts of IL-12p70 without compromising the ability of MDDC to migrate in response to CCR7 stimulation in vitro. These investigators reported that MDDC transfected with IL-12p70 induced an antigen-specific response of larger magnitude than those transfected without IL-12p70; however, they did not examine the cytokine profiles of the induced T-cell lines.

In the experiments we present here, we investigated the effect that IL-12p70 transfection had on activating the innate immune system and on the phenotype of induced T-cell responses. IL-12p70–transfected MDDC were shown to produce significant quantities of IL-12p70 (1–4 ng/106 cells/d; Fig. 1). The majority of IL-12 was secreted within the first 6 hours following transfection, and production of IL-12 was retained even after MDDC were immediately frozen and thawed. Several studies have indicated that the migration of DC into tumor-draining lymph nodes occurs as early as 0.5 to 6 hours following injection intradermally (4244). Our studies suggest that IL-12p70–transfected DC that are trafficking to draining lymph nodes would still be secreting sufficient amounts of IL-12 to skew naïve T cells to a helper-type phenotype. Moreover, various strategies, such as using ARCA-capped mRNA and devising mRNA stability configurations that increase the half-life and translation efficiency of the transfected mRNA, would be likely to improve the production and, possibly, duration of IL-12 production.

IL-12p70–transfected MDDC enhanced NK activation in overnight assays as well as NK cell cytolytic activity, specifically against K562 target cells but not autologous MDDC (Fig. 2). Transfection with IL-12p70 also altered the cytokine profiles of allogeneic antigen-specific T-cell lines that expanded in response to transfected MDDC. Whereas immature or cytokine cocktail–matured DC reproducibly induced TH2-skewed T cells, this phenotype was reversed when the DC were transfected with IL-12p70, resulting in a significant increase in the ratio of TNF-α/IL-5 produced in response to allogeneic targets (Fig. 3). Additionally, we compared LPS- and poly I:C–matured MDDC to MDDC matured with cytokine cocktail in their ability to skew CD4+ T-cell responses to TH1. Only MDDC matured with poly I:C were capable of secreting IL-12p70. As anticipated, poly I:C–matured, but not LPS-matured, MDDC inhibited the priming of TH2 type T cells in the allogeneic mixed lymphocyte reaction (Fig. 3D). Poly I:C has not as yet been proved to be an effective DC maturation stimulus for use in vivo, whereas cytokine cocktail containing PGE2 is a widely used and Food and Drug Administration–approved method for preparing vaccine grade MDDC. IL-12p70 mRNA transfection of cytokine cocktail–matured MDDC represents a new approach to improve the quality of cells delivered in vivo.

Our studies revealed that IL-12p70 transfection enhanced the antigen specificity and the TH1 type skewing of T-cell lines induced from PBMC of healthy donors and of melanoma patients by autologous MART-1–transfected MDDC (Figs. 3, 5, and 6). Consistent with several studies examining the induction of TH2 responses of vaccine-induced immune responses in melanoma (9, 10), we found that repeated rounds of stimulation with autologous cytokine cocktail–matured MDDC led to an overall increase in IL-5 production of the T cells that were primed. However, the transfection of the MDDC with IL-12 significantly inhibited IL-5 production and conversely up-regulated the production of IFN-γ by the T cells. Although a large emphasis has been placed on the role of CD4+ T helper cells in establishing the TH1/TH2 balance, the role of CD8+ T cells in balancing the dichotomy of the type 1/type 2 (Tc1/Tc2) immune response has been described to a lesser extent (4547). Several studies have illustrated the capability of CD8+ T cells to develop into type 2, Tc2 cells, capable of secreting “type 2” cytokines, including IL-4 and IL-5, with decreased antitumor capacity (4850). Our study showed that both CD4+ and CD8+ T cells were shifted to a “type 1” phenotype by IL-12 transfection of DC. The specificity of the immune response generated in the T-cell priming cultures of the melanoma patients was predominantly toward the immunodominant region of MART-1 [i.e., MART(21–35)]. Consistently, these responses were mostly of CD8+ T cells. The in vitro priming conditions may allow for favored priming of the CD8+ T cells due to the high frequency of precursor CD8+ T cells toward this epitope in HLA-A*0201 individuals. Nonetheless, a highly type 1 skewed immune response was elicited in CD8+ T cells by priming with IL-12–transfected DC. Taken together, these results show that transfection with IL-12p70 mRNA can enhance antitumor T-cell responses not only quantitatively, in terms of the magnitude, but also qualitatively, in terms of phenotype.

Many previous studies have established the promise of DC vaccines as a potentially effective mode of therapy for cancer patients. IL-12p70 could play an important role in the development of beneficial antitumor immunity. However, for recombinant IL-12p70 to be delivered by injection, there is a risk that the minimal effective systemic dose would be accompanied by severe toxicity. MDDC that are capable of migrating to the lymph node and secreting IL-12p70 may provide a very potent vehicle for delivery of IL-12p70 to naïve T cells in the lymph node without a risk of severe systemic side effects. Our results show the potential usefulness of IL-12p70–transfected MDDC derived from the blood of melanoma patients for the induction of innate and adaptive immune responses that exhibit a strong proinflammatory phenotype and can aid in the reversal of type 2 skewing of the antitumor immune response evoked in melanoma patients.

N. Bhardwaj: co-inventor on patents related to human DC production. R.T. Gandhi: consultant/advisory board, Pfizer; educational grants from Tibotec and Boehringer-Ingelheim.

Note: Supplementary data for this article are available at Cancer Research Online (http://cancerres.aacrjournals.org/).

Grant support: NIH grants F32 AI058457-03 (D.G. Kavanagh), R01 AI066992-01A1 (R.T. Gandhi), R01 044628 (N. Bhardwaj), and R01 1061684 (N. Bhardwaj); the Doris Duke, Emerald, and Burroughs Wellcome Foundations and the Elizabeth Glaser Pediatric AIDS Foundation (N. Bhardwaj); and the Howard Hughes Medical Institute (M.A. Brockman and B.D. Walker).

The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

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Supplementary data