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Immunology and Infection

Preparation of Tumor Antigen-loaded Mature Dendritic Cells for Immunotherapy

Published: August 1st, 2013

DOI:

10.3791/50085

1Cancer Institute, NYU Langone Medical Center, 2Infectious Diseases, NYU Langone Medical Center

The most commonly used method for generating large numbers of autologous dendritic cells (DCs) for use in tumor immunotherapy is described. The method uses IL-4 and GM-CSF to differentiate DCs from monocytes. The immature DCs are stimulated to mature and then loaded with antigens before they are injected back into the patient.

While clinical studies have established that antigen-loaded DC vaccines are safe and promising therapy for tumors 1, their clinical efficacy remains to be established. The method described below, prepared in accordance with Good Manufacturing Process (GMP) guidelines, is an optimization of the most common ex vivo preparation method for generating large numbers of DCs for clinical studies 2.

Our method utilizes the synthetic TLR 3 agonist Polyinosinic-Polycytidylic Acid-poly-L-lysine Carboxymethylcellulose (Poly-ICLC) to stimulate the DCs. Our previous study established that Poly-ICLC is the most potent individual maturation stimulus for human DCs as assessed by an upregulation of CD83 and CD86, induction of interleukin-12 (IL-12), tumor necrosis factor (TNF), interferon gamma-induced protein 10 (IP-10), interleukmin 1 (IL-1), and type I interferons (IFN), and minimal interleukin 10 (IL-10) production.

DCs are differentiated from frozen peripheral blood mononuclear cells (PBMCs) obtained by leukapheresis. PBMCs are isolated by Ficoll gradient centrifugation and frozen in aliquots. On Day 1, PBMCs are thawed and plated onto tissue culture flasks to select for monocytes which adhere to the plastic surface after 1-2 hr incubation at 37 °C in the tissue culture incubator. After incubation, the lymphocytes are washed off and the adherent monocytes are cultured for 5 days in the presence of interleukin-4 (IL-4) and granulocyte macrophage-colony stimulating factor (GM-CSF) to differentiate to immature DCs. On Day 6, immature DCs are pulsed with the keyhole limpet hemocyanin (KLH) protein which serves as a control for the quality of the vaccine and may boost the immunogenicity of the vaccine 3. The DCs are stimulated to mature, loaded with peptide antigens, and incubated overnight. On Day 7, the cells are washed, and frozen in 1 ml aliquots containing 4 - 20 x 106 cells using a controlled-rate freezer. Lot release testing for the batches of DCs is performed and must meet minimum specifications before they are injected into patients.

1. Isolation and Cryopreservation of PBMCs 4

  1. Aseptically spike one of the access ports in the leukapheresis bag using a plasma transfer set. Using a 60 ml syringe, transfer the leukapheresis obtained from patients into a sterile 500 ml bottle.
  2. Adjust the volume of the leukapheresis to 2x its original volume using room temperature RPMI. Mix thoroughly.
  3. Gently mix the bottle of Ficoll-Paque PLUS. Add 12 ml Ficoll-Paque PLUS into sterile 50 ml conical tube.
  4. Gently layer 3.......

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Between 10 - 20% of starting PBMCs differentiate into DCs at the end of the culture period. Mature DCs are CD11c+, CD14-, CD83+, CD40+, and CCR7+ (Figure 1). They express high levels of MHC class I and II molecules and the costimulatory molecules CD80 and CD86. Poly-ICLC also induced lower levels of PDL-1 as compared to other TLR agonists 14. Additionally, these Poly-IC-matured DCs secrete large amounts of IL-12 (Figure 2 and 15,16) and induce the proliferation of a.......

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Phase I and II clinical trials of monocyte-derived DCs have shown that they induce immune responses in patients however clinical success has been limited 1. This may be partly due to the lack of consensus on how to generate the optimal DCs for tumor immunotherapeutic use. Although there are numerous ways to generate clinical-grade DCs, these methods vary in terms of the use of cytokines used to differentiate the monocytes, stimuli used to induce maturation, and methods of antigen loading. The formula for gener.......

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The authors would like to thank Andres Salazar (Oncovir, Inc.) for the gift of the Poly-ICLC.

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Name Company Catalog Number Comments
Reagent/Supplies/Equipment Manufacturer Catalog No.
RPMI-1640 medium with L-glutamine BioWhittaker 12-702F
1M HEPES buffered saline BioWhittaker 17-737E
Phosphate buffered saline (PBS) BioWhittaker 17-516F
Human albumin, 25% solution USP Aventis Behring
Ficoll-Hypaque PREMIUM GE Healthcare 17-5442-03
Human AB serum Valley Biomedical HP1022
Sterile saline USP Hospira
CryoMACS DMSO Miltenyi Biotec 170-076-303
Leukine GM-CSF, 0.5 mg/ml Berlex A02266
MACS GMP IL-4 Miltenyi Biotec 170-076-101
Hiltonol, Poly-ICLC, 2 mg/ml Oncovir NA
VACMUNE KLH Biosyn
225 sq cm EasyFlasks Nalgene Nunc 159934
Falcon 6-well tissue culture plates Becton Dickinson 353046
1.8 ml CryoTube vials Nalgene Nunc 377267
Controlled Rate Freezer Thermo CryoMed

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