|
|
Blood, Vol. 114, Issue 9, 1736-1745, August 27, 2009
Granulocyte-macrophage colony-stimulating factor (GM-CSF)–secreting cellular immunotherapy in combination with autologous stem cell transplantation (ASCT) as postremission therapy for acute myeloid leukemia (AML)
Blood Borrello et al.
114: 1736
Supplemental materials for: Borrello et al
Quantitative analysis of WT1 transcipt levels: White blood cells were extracted from anti-coagulated bone marrow and/or peripheral blood samples by hypotonic RBC lysis. Total RNA was extracted using RNA STAT-60 (Tel-Test, Friendswood, TX). One to five micrograms of total RNA was synthesized into cDNA according to standard procedures in the SuperScript™ Preamplification System (Life Technologies/Invitrogen, Rockville, MD). Patient specimens, serial six-fold dilutions of K562 cell line cDNA (0.08–250 ng/µl), and no template controls were amplified in triplicate according to methods described in the TaqMan™ PCR Core Reagent Kit (Perkin Elmer, Foster City, CA). WT-1 expression levels were detected using a transcript specific primer and probe set. The probe was labeled with a 5′ reporter dye, 6-carboxy-fluorescein (FAM), and a 3′ quencher dye, 6-carboxy-tetramethyl-rhodamine (TAMRA). All amplification reactions contained 1 µl of cDNA in a total volume of 20 µl. Similarly, the ABL gene was amplified in order to determine unknown quantities of RNA and to quantitate WT1 transcript copy number relative to an endogenous control. The amplification conditions for both the WT1 and ABL amplifications were the following: 10 minutes at 95°C (1 cycle); 15 seconds at 95°C and 1 minute at 60°C (40 cycles). The absolute sensitivity of the assay was determined from limiting serial dilutions of K562 cDNA in normal bone marrow cDNA and was found to be between 10−6 and 10−7. Following PCR amplification of WT1 and ABL transcripts, all patient and negative control CT (threshold cycle) values were compared to their respective standard curves. Absolute transcript copy number was determined for each reaction and an average transcript copy number was determined for all triplicates. In order to compensate for differences in RNA integrity and cDNA synthesis efficiency, the absolute WT1 transcript copy number was normalized to the endogenous control gene, ABL. The limit of normalized WT1 transcript quantification was 10−3 WT1/ABL. Elispot analysis of in vitro T-cell immune response: One vial of autologous leukemia blasts collected at initial diagnosis was thawed along with one vial of PBMCs from each timepoint. Cells were rested in complete medium (1ml AIM-V+10% human AB serum per well in 48 well plates) for one hour at 37°C, filtered through a nylon cell strainer to remove debris, ficolled and washed in fresh media. Leukemia cells were irradated with 4000 cGy and plated with PBMCs at the following density in 48 well plates: 2 × 106 PBMC to 5 × 105 tumor cell for 7 days. On day 7, all cells were harvested from wells and counted. A fresh vial of autologous leukemia blasts was thawed, rested as above for 1 hour and washed before being plated in IFN-γ and Granzyme B Elispot assays at a density of 1 × 105 T cells per well at a T cell to tumor cell ratio of 2:1. ELISPOT: IFN-γ and Granzyme B ELISPOT assays: MultiScreen-IP PVDF plates (Millipore, Bedford, MA) were pre-wetted with 70% ethanol, washed, and coated overnight at 4°C with 1 µg per well of anti-human IFN-γ antibody 1-D1K (Mabtech, Mariemont, OH) or anti-human Granzyme B clone GB10 (Mabech, Mariemont, OH) in PBS. The plates were washed and blocked with culture medium, RMPI (Cambrex, Walkersville, MD) with 10% human AB serum (Gemini Bio-Products, Woodland, CA), for 2 hrs at 37°C. Cells from the 7 day stimulation cultures were plated in triplicates at 1 × 105 cells/well/0.1 ml with autologous leukemic blasts. T cells were also incubated with a control CEF peptide pool (Mabtech) at 2µg/ml. After 20-hr incubation at 37°C, cells were removed by 6 washes with PBS/0.05% Tween 20 followed by 2 washes with PBS alone. Captured IFN-γ was detected by the addition of 200 ng/well of biotinylated anti-human IFN-γ antibody 7-B6-1 (Mabtech) followed by 2-hr incubation at 37°C. Captured Granzyme B was detected by the addition of 200 ng/well of biotinylated anti-human Granzyme B antibody GB11 (Mabtech) followed by 2-hr incubation at 37°C. After washing, as above, avidin-peroxidase complex (Vectastain Elite ABC kit, Vector Laboratories, Burlingame, CA) was added for 1 hr at room temperature. Plates were washed again 8 times as above, and peroxidase staining was performed with AEC (Sigma, St. Louis, MO) substrate in acetate buffer for about 5 min. The reaction was stopped by rinsing the plates under running distilled water. After drying overnight, spot numbers were evaluated on KS ELISPOT Automated Reader System with KS ELISPOT software (Zeiss, Hallbergmoos, Germany). Mean numbers of spot forming cells (SFC) from triplicate wells were calculated and expressed as spots per 1 × 106 PBMC. Mean spot numbers from wells with PBMCs incubated with medium alone (background) were subtracted from means of PBMCs stimulated with antigens. Statistical analysis of differences between samples was performed using a paired student t-test.
Files in this Data Supplement:
|
|
