Interleukin 4 (il 4)

Monocyte-derived DCs were established from adherent peripheral blood mononuclear cells (PBMC) obtained via leukapheresis performed at the UCLA Hemapheresis Unit, as we have published previously^{3 (link),6 (link),52 (link)}. All ex vivo DC preparations were performed in the UCLA-Jonsson Cancer Center GMP facility under sterile and monitored conditions. In brief, dendritic cells were prepared by culturing adherent cells from peripheral blood in RPMI-1640 (Gibco) and supplemented with 10% autologous serum, 500 U/mL GM-CSF (Leukine®, Amgen, Thousand Oaks, CA) and 500 U/mL of IL-4 (CellGenix), using techniques described previously^{2 (link)}. Following culture, DCs were collected by vigorous rinsing and washed with sterile 0.9% NaCl solution. The purity and phenotype of each DC lot was also determined by flow cytometry (FACScan flow cytometer; BD Biosciences, San Jose, CA). Cells were stained with FITC-conjugated CD83, PE-conjugated CD86 and PerCP-conjugated HLA-DR mAb’s (BD Biosciences). Release criteria were >70% viable by trypan blue exclusion, and >30% of the large cell gate being CD86⁺ and HLA-DR⁺. One day before each vaccination, DC were pulsed (co-cultured) with tumor lysate overnight, washed, and the final product was tested for sterility by Gram stain, mycoplasma, and endotoxin testing prior to injection.

PBMCs were collected using a ficoll gradient (Ficoll-Paque Plus, GE Healthcare) and monocytes were isolated by a CD14 positive selection (Miltenyi, Bergisch Gladbach, Germany). Monocytes were split into five experimental groups: (1) Negative Control (no cytokines), (2) GM–CSF (Sanofi) + IL-4 (Cell Genix) at 1000 U/ml, 3) Recombinant IL32α (R&D Systems) at 100 ng/ml, 4) Recombinant IL32β (R&D Systems) at 100 ng/ml, 5) Recombinant IL32γ (R&D Systems) at 100 ng/ml, and cultured using Cell Genix Media to yield immature DCs at day 5. Immature DC were harvested and surface stained for flow cytometry analysis. Cell surface markers were observed on the double positive, HLA-DR and CD86 population of cells. Antibodies used included CD80 FITC (BD, Clone L307.4), Mouse IgG1 FITC (Beckman Coulter PN IM0639U), CD86 Pe-Cy7 (BD, Clone FUN-1), HLA-DR PerCpCy5.5 (BD, Clone G46-4), CD1B APC (BioLegend, Clone SN13), CD14 APC-Cy 7 (BD, Clone MφP9), CD68 BV 711 (BD, Clone Y1/82A), Mouse IgG2B BV 711 (BD, Clone 27-35), and Zombie Aqua Viability Dye BV 510 (BioLegend).

Primary human pDC and monocytes were isolated from buffy coats of healthy blood donors provided by the Blutbank Springe (Germany) using ficoll density gradient centrifugation and subsequent magnetic activated cell sorting (Diamond Plasmacytoid Dendritic Cell Isolation Kit, CD14⁺ Cell Isolation Kit; Miltenyi Biotec). Following isolation, 2 x 10⁵ pDC were cultivated for 1 h in 200 μl of 10 ng/ml interleukin 3 containing serum-free DC medium (CellGenix) and were then treated as indicated. moDC, GM-CSF MΦ, and M-CSF MΦ were differentiated from 5 x 10⁵/500 μl monocytes for 5 days in serum-free DC medium enriched with 1000 U/ml GM-CSF (granulocyte macrophage-colony stimulating factor, CellGenix) and 1000 U/ml IL-4 (CellGenix), or 80 U/ml GM-CSF, or 100 ng/ml M-CSF (macrophage-colony stimulating factor, Miltenyi Biotec), respectively.

Monocytes were enriched from leukapheresis products as described before [25 (link)]. Monocytes were cultured in X-VIVO 15 medium (Lonza) supplemented with 2% human serum (HS; Sanquin), IL-4 (500 U/ml), GM-CSF (800 U/ml, both CellGenix) and KLH (10 μg/ml, Calbiochem). DCs were matured with a cocktail of 10 ng/ml TNF-α, 5 ng/ml IL-1β, 15 ng/ml IL-6 (all CellGenix) and prostaglandin E₂ (10 μg/ml, Pharmacia & Upjohn) [26 (link)]. Cells used for the DTH skin test were cultured without KLH. DCs were electroporated with mRNA encoding gp100 or tyrosinase, as previously described [27 (link)]. Patients could only participate if the predefined phenotypic minimal release criteria used in clinical trials were met [28 (link)]. DCs were administered both intradermally (maximum of 10 × 10⁶ cells) and intravenously (maximum of 20 × 10⁶ cells).