IL-12p70-producing patient DC vaccine elicits Tc1-polarized immunity

Abstract

Background: Systemic administration of IL-12p70 has demonstrated clinical activity in cancer patients, but dose-limiting toxicities have hindered its incorporation in vaccine formulations. Here, we report on the immunological and clinical outcomes upon vaccination with CD40L/IFN-γ-matured, IL-12p70-producing DCs.

Methods: 7 HLA-A*0201+ newly diagnosed stage IV melanoma patients were immunized against the gp100 melanoma antigen using autologous peptide-pulsed, CD40L/IFN-γ-matured DCs. PBMCs were taken weekly for immune monitoring by tetramer analysis and functional assays. CT imaging was performed at baseline, week 9, and week 18 for clinical assessment using RECIST.

Results: 6 of 7 treated patients developed sustained T cell immunity to all 3 melanoma gp100 antigen-derived peptides. 3 of the 6 immunological responders developed confirmed clinical responses (1 complete remission >4 years, 2 partial response). Importantly, DC vaccine-derived IL-12p70 levels positively correlated with time to progression (P = 0.019, log-rank), as did T-cytotoxic 1 (Tc1) immunity, as assessed by IFN-γ/IL-13 and IFN-γ/IL-5 ratios (P = 0.035 and P = 0.030, respectively, log-rank). In contrast, a pathway-specific defect in IL-12p35 transcription was identified upon CD40L/IFN-γ activation in clinical nonresponder patient DCs, and gp100-specific T cells from these patients displayed a Tc2 phenotype. Incorporation of TLR3 and TLR8 agonists into the CD40L/IFN-γ activation protocol corrected the IL-12p70 production defect in DCs derived from clinical nonresponder patients.

Conclusion: These findings underscore the essential role of IL-12p70 in the development of therapeutic type 1 antigen-specific CD8+ T cell immunity in humans with cancer.

Trial registration: ClinicalTrials.gov NCT00683670.

Figures

Figure 1. Clinical trial scheme and ex vivo IL-12p70 production by CD40L/IFN-γ–activated DCs (mDCs).

(A) Consort diagram. (B) Study timelines depicting cyclophosphamide treatment (300 mg/m2 i.v.), DC vaccinations (D1–D6), and PBMC sampling for immune monitoring. (C) Ex vivo IL-12p70 levels produced by patient-derived mDCs used for manufacturing D1–D3 (each symbol represent a vaccine dose). DC supernatants were harvested 24 hours after activation, and IL-12p70 concentrations were determined by ELISA. D1 was manufactured using fresh PBMCs (which consistently produced the highest concentration of cytokine) as the source of monocytes, and D2–D6 were prepared using cryopreserved PBMCs. Results represent mean ± SEM.

Figure 2. Vaccine-induced CD8+ T cell responses.

PBMCs isolated before DC vaccination (pre-vacc) and at peak (post-vacc; see Supplemental Figure 4 for kinetics) were cultured in vitro in the presence of peptide (40 μg/ml) and IL-2 (50 U/ml) for 12 days. Cultures were stained by addition of corresponding PE-conjugated tetramers (gp100 peptide/HLA-A*0201) followed by a lineage-negative cocktail (FITC-conjugated anti-CD4, -CD14, -CD19, and -CD56) and allophycocyanin-conjugated anti-CD8. Viability dye 7AAD was added to gate out dead cells. Numbers within dot plots represent percent G154 (A), G209-2M (B), and G280-9V (C) tetramer-positive cells in lymph+/CD8+ gated cells.

Figure 3. Vaccine-induced T cells recognize native gp100 and display high affinity for antigen.

(A) PBMCs collected after D3 were cultured in the presence of G209-2M (white bars and symbols) or G280-9V (black bars and symbols) peptide for 12 days as described in Figure 2 and tested in a standard 4-hour 51Cr release assay for their ability to recognize native gp100 antigen on human melanoma cells lines DM6 (HLA-A2+gp100+) and A375 (HLA-A2+gp100–). Percent specific lysis at a 30:1 effector/target ratio is shown; spontaneous lysis was <10%. Results are representative of 2 experiments. (B) Avidity of G209-2M– and G280-9V–specific T cells was determined in a standard 4-hour 51Cr release assay using peptide titrations and T2 (HLA-A*0201+) cells as targets. Percent specific lysis is shown for each peptide concentration; spontaneous lysis was <5%. Results are representative of 2 experiments.

Figure 4. IL-12p70 production and TTP.

Cox regression analysis followed by likelihood-ratio test revealed a positive correlation between IL-12p70 production and TTP (P = 0.0198, log-rank). White symbols represent patients with rapid disease progression. Black symbols represent patients with confirmed clinical response (see Table 1). P1 showed complete remission as of 4 years after initiation of treatment (analysis performed on 08/05/12; asterisk); P5 and P6 showed a partial response, with disease progression observed at or after 11.5 months of treatment initiation. No correlation was observed between IL-12p70 production and immune response or between immune response and clinical outcome.

Figure 5. gp100 antigen–specific T cells from clinical responders display a Tc1 phenotype.

Purified CD8+ T cells were stimulated twice in vitro, and antigen-specific frequencies were determined by peptide/HLA-A*0201 tetramers. T cells were adjusted to 106 cell/ml and stimulated with antigen, and supernatants were harvested at 20 hours. Cytokine production was determined as described in Methods. (A and B) Patients were grouped as clinical responders (A) and nonresponders (B). To compare production of Tc1 (IFN-γ) and Tc2 (IL-5, IL-13) cytokines among patients, a cytokine index was derived by dividing pg/ml IFN-γ by pg/ml IL-13 or IL-5. (C) Cytokine ratios differed among clinical responder and nonresponder patients. P values (unpaired 2-tailed t test) are indicated. Results are representative of 2 experiments.

Figure 6. IL-12p70 production and IL-12p35 transcription by mDCs derived from patients with melanoma.

(A) DCs from age- and gender-matched healthy donors (H) and melanoma patients (M) were activated with CD40L/IFN-γ for 24 hours, and supernatants were harvested and assayed for IL-12p70 production by ELISA. Horizontal lines and error bars denote median and interquartile range. P value (Wilcoxon matched-pairs test) is indicated. (B) Patient DCs were activated with CD40L/IFN-γ for 24 hours, supernatants were collected, and IL-12p40 and IL-12p70 production was measured by ELISA. Results are shown for 10 melanoma patients. Horizontal lines and error bars denote median and interquartile range. (C) To examine IL-12p35 gene activation, DCs were activated with CD40L/IFN-γ for 6 hours, cells were harvested and washed, and total RNA was prepared. Total RNA was also prepared from iDCs. Using primers specific for IL-12p35 and CD11c (DC lineage marker), qRT-PCR was performed and analyzed using the relative standard method. Results were normalized to CD11c, and values are expressed as fold IL-12p35 induction in mDCs relative to iDCs. Results are representative of 2 experiments.

Figure 7. IL-12p70 production and ex vivo antigen-specific T cell responses stimulated by CD40L/IFN-γ + poly I:C + R848–activated DCs.

(A) iDCs were stimulated for 24 hours with CD40L/IFN-γ (mDCs), alone or in combination with poly I:C (5 μg/ml; TLR3 agonist) and R848 (5 μg/ml; TLR8 agonist), and supernatants were assayed for IL-12p70. Results are those obtained in 2 experiments. P values (paired 2-tailed t test) are indicated. (B and C) DCs derived from P3 and P4 were activated for 24 hours with CD40L/IFN-γ, alone or in combination with poly I:C and R848, and supernatants were analyzed for IL-12p70. Additionally, activated DCs were pulsed with G280-9V peptide (40 μg/ml) and used for stimulation of purified CD8+ T cells (see Methods). G280-9V–specific T cell frequencies within the CD8+ T cell population was determined at day 12 using YLE/HLA-A*0201 tetramers and anti-CD8 mAb. Results are representative of 3 experiments.