Cell lines and peptides
All tumor cell lines were obtained from ATCC and from cell line services (Eppelheim, Germany) and grown in RPMI 1640 medium supplemented with 10% fetal calf serum, 2 mmol/L L-glutamine and antibiotics. Tissue culture media and supplements were purchased from PAA (Cölbe, Germany) unless indicated otherwise.
Peptides were obtained from the Peptide Synthesis Facility at the DKFZ. They were dissolved in DMSO and further diluted in PBS. Peptides spanned the entire frameshift-sequence of Caspase-5 (-1) (40 mer; containing 25 aa neo- and 15 aa wt-sequence), OGT (-1) (34 mer; 19 aa neo-, 15 aa wt), Sec63 (-1) (42 mer; 27 aa neo-. 15 aa wt), TGFβRII (-1) (49 mer; 34 aa neo-, 15 aa wt), Taf1B (-1) (40 mer; 25 aa neo-, 15 aa wt), HT001 (-1) ( 47 mer; 32 aa neo-, 15 aa wt), MSH-3 (-1) (40 mer; 31 aa neo-, 9 aa wt) and AIM2 (-1) (20 mer; 13 aa neo-, 7 aa wt). Overlapping 20 mer peptides were synthesized for U79260 (-1) (49 aa neo-, 1 aa wt); AC-1 (-1) (58 aa neo-, 2 aa wt); FLJ11053 (-1) (32 aa neo-, 9 aa wt) together with FLJ378 (-2) (37 aa neo-, 9 aa wt); DAMS (-1) (14 aa neo-, 9 aa wt) together with DAMS (-2) (30 aa neo-, 9 aa wt) and UST3 (-1) (66 aa neo-, 4 aa wt) respectively (for peptide sequences see ).
B cell and T cell purification
Peripheral blood mononuclear cells were isolated from heparinized blood of a healthy HLA-A02+ donor using Ficoll-density gradient centrifugation. Whole CD3+ T cells were obtained from PBMCs by magnetic depletion of non-T cells using the MACS Pan T Cell Isolation Kit II (Miltenyi; Bergisch Gladbach, Germany) according to manufacturer's instructions. The remaining non-T cells were subsequently used as a source for B cells. All procedures using human cells were approved by the Ethics Committee of the University of Heidelberg in accordance with the provisions of the declaration of Helsinki (as revised in Edinburgh 2000). An informed consent in written was obtained prior to the blood sampling procedure.
Culture of CD40 Bs
The culture of CD40 Bs was performed as described . Briefly, B cells were stimulated via NIH3T3 stimulator cells stably expressing human CD154. Lethally irradiated stimulator cells (60 Gy) were plated on 6-well plates (0.35 × 105 cells/well) and cultured overnight. After removing medium, magnetic depleted non Tc (2 × 106 cells/ml) were resuspended in Iscove's modified DMEM (IMDM) supplemented with 10% human AB serum, 5 μg/ml insulin, 50 μg/ml transferrin and 15 μg/ml gentamicin in the presence of IL-4 (10 IU/ml) and cyclosporin A (5.5 × 10-7M). Every 3-5 days cells were transferred to new plates containing fresh irradiated stimulator cells. All cytokines were obtained from PromoCell, Heidelberg, Germany.
Fusion of MSI+ tumor cells with CD40 Bs
HCT116 tumor cells and CD40 Bs from a healthy HLA-A02+ donor were washed in serum-free RPMI 1640 medium, pelleted and stained with 5 μM 5-chloromethylfluorescein diacetate or 5 μM 5-(and 6)-(((4-chloromethyl) benzoyl)-amino) tetramethyl-rhodamin in PBS, respectively. Cells were incubated for 45 min at 37°C, washed and resuspended in serum-free RPMI 1640 medium. After a second incubation period, stained cells were resuspended in serum-free RPMI 1640. CD40 Bs were mixed with tumor cells at a CD40 Bs:tumor cell ratio of 2:1 and exposed 5 min to 4 × 10-5M SDS. Cell suspension was pelleted and 1 ml 50% polyethylene glycol 1500 (Roche, Mannheim, Germany) was added over 5 min to the CD40 Bs/tumor cell pellet while stirring the cells continuously. The polyethylene glycol solution was then diluted by slow addition of first 1 ml warmed serum- and phenol red-free medium over 3 min with continued stirring and then another 10 ml of this medium over 5 min. After centrifugation cells were resuspended in phenol red-free RPMI 1640 supplemented with 10% FCS. Cells were cultured 24 h before the fusion efficacy was analyzed by flow cytometry. Fused cells consisting of MSI+ colon carcinoma cells HCT116 and CD40 Bs, which exhibited dual fluorescence, were cloned by limiting dilution. Briefly, fused cells were seeded under limiting dilution conditions (0.7 cells/well) in 96-well plates containing a final volume of 200 μl RPMI 1640 with 10% FCS. Outgrowing fusion clones were comprehensively screened concerning the cell surface expression of HLA-A02, MHC class I and II molecules and costimulators (CD40, CD80, and CD86). For all subsequent experiments, two clones were selected and designated as Fc1 and Fc2.
T cell stimulation with semiallogenic cellular fusion clones
Fusion clones were collected from cell culture, irradiated (200 Gy) and added to purified CD3+ autologous T cells at a ratio of 3:1 (Tc:fusion cell) in IMDM supplemented with 10% human AB serum, 5 μg/ml insulin, 50 μg/ml transferrin and 15 μg/ml gentamicin in the presence of IL-7 (10 IU/ml ). Cells were plated at a density of 2 × 106 T cells in 1 ml of medium per well of a 24 well plate. For T cell restimulation this was repeated weekly. IL-2 was first given at day 21 (10 IU/ml), also at day 24 and from day 28 on only IL-2 was used instead of IL-7.
CD4+ T cells and CD8+ T cells were obtained from the whole T cell population by magnetic cell sorting using MACS CD4 and CD8 microbeads (Miltenyi; Bergisch Gladbach, Germany) according to manufacturer's instructions. CD4 microbeads were used for the sorting of CD8+ T cells and CD8 microbeads for the sorting of CD4+ T cells. Selection of T cells was checked by flow cytometry. CD4+ T cells and CD8+ T cells were restimulated separately with irradiated fusion clones and IL-2 as described for the whole T cell population.
IFN-γ ELISpot assay
ELISpot assays were performed using nitrocellulose-96-well plates (Multiscreen; Millipore, Bedford, USA) covered with mouse anti-human IFN-γ mAb (Mabtech, Sweden) and blocked with serum containing medium. Initially, in vitro primed T cells (1 × 104) were stimulated in triplicates with 2 × 104 CD40 Bs, HCT116, Fc1 or Fc2 cells per well as targets. Afterwards T effector cells (1 × 104) were plated in sixplicates with 2 × 104 peptide-loaded autologous CD40 Bs. Peptides were added at a final concentration of 10 μg/ml. After incubation for 16 h at 37°C, plates were washed, incubated with biotinylated rabbit anti-human IFN-γ for 4 h, washed again, incubated with streptavidin-alkaline phosphatase for 2 h, followed by a final wash. Spots were detected by incubation with NBT/BCIP (Sigma-Aldrich, Steinheim, Germany) up to 1 h. Reaction was stopped with water and after drying spots were counted. The deduced frequency of peptide-specific T cells was calculated by subtracting mean numbers of spots in the no-peptide control from mean numbers of spots in the peptide-stimulated sample. Negative values were scored as zero.
After proving the assumption of normality, differences between negative control and FSP-containing wells were determined by using the unpaired Student's t-test. If normality failed, the nonparametric Mann-Whitney U-Test was applied. The tests were performed by using Sigma-Stat 3.0 (Jandel Corp, San Rafael, CA). The criterion for significance was set to p < 0.05.
Determination of cMS mutation pattern
Genomic DNA was isolated from microdissected tumor sections using the DNeasy Tissue Kit (Qiagen, Hilden, Germany). For analysis of cMS, the corresponding genomic regions were amplified as described previously . PCR products were analyzed using an ABI3100 genetic analyzer and Genescan Analysis Software (Applied Biosystems, Darmstadt, Germany). Instability was scored, if comparison with amplification products of normal tissue revealed the occurrence of novel peaks or if the ratio of corresponding peak areas was ≤0.5 or ≥2.
Standard chromium release assays were performed as described . Tumor target cells were labelled with 100 μCi [51Cr]-sodium chromate for one hour at 37°C. For each experimental condition, cells were plated in V-bottomed 96-well plates with 103 target cells/well in triplicate. Varying numbers of CTL were added to a final volume of 200 μl and incubated for 4 h at 37°C. Spontaneous and maximal release was determined in the presence of medium alone or of 1% NP-40. Supernatants (100 μl/well) were harvested and counted in a gamma-counter. The percentage of specific lysis was calculated as follows: 100% × [experimental release - spontaneous release]/[maximal release - spontaneous release].
Expression of the following surface markers was analyzed: MHCI (W6/32), HLA-A2 (BB7.2), MHCII (12G6), CD3 (OKT3), CD28 (CD28.2) with unlabeled primary antibodies and FITC-labelled goat anti-mouse IgG's (Dianova, Hamburg, Germany) as second antibody. Cells treated without primary antibody were used as negative control. CD4 (RPA-T4), CD8 (RPA-T8), CD19 (HIB19), CD45RO (UCHL1), CD50 (101-1D2, Cymbus Biotechnology, UK), CD58 (1C3 AICD58.6), CD80 (BB1/B7-1), CD86 (B70/B7-2) and CD102 (B-T1; Serotec, Oxford, UK) were directly FITC-conjugated, whereas CD25 (M-A251), CD69 (FN50) and CD71 (SOM4D10, Diatec, Oslo, Sweden) were PE-conjugated. Isotype-matched monoclonal antibodies were used as a negative control.
For intracellular staining, T cells were incubated with the protein transport inhibitor brefeldin A (2 μg/ml, Serva, Deisenhofen, Germany) for 15 h at 37°C. After two washes with PBS/1% FCS, cells were fixed with cold 4% paraformaldehyde (PFA, Serva) in PBS for 10 min at 4°C, washed twice with PBS/1% FCS and permeabilized with saponine buffer (PBS, 0,1% saponine, 1% FCS and 0,01 M Hepes) for 10 min at room temperature. T cells were subsequently stained for intracellular cytokines with PE-labelled anti-IFN-γ (4SB3), anti-TNF-α(Mab11), anti-IL-2 (MQ1-17H12) mAbs, or IgG1 isotype control mAb in saponine buffer for 20 min at 4°C. Cells were washed twice with saponine buffer and resuspended in PBS/1% FCS before measurement. Intracellular perforin was detected with PE-labelled anti-perforin (dG9) mAb. For analysis of intracellular granzyme B, cells were incubated with anti-granzyme B (2C5/F5, Serotec) mAb followed by FITC-labelled goat anti-mouse IgG's. Cells treated without primary antibody were used as negative control. Cell surface and intracellular immunofluorescences were obtained using FACSCalibur (BD Biosciences, Heidelberg, Germany) and CellQuest software. Typically, 20.000 cells were acquired from each sample. Cells to be analyzed were first gated according to reasonable size and granularity in the forward/sideward scatter plot. Gated cells were than blotted into histogram blots. Mean intensities of the negative control were between 2 and 10. For determination of percentages of positive cells, the cut-off included a maximum of 2% of the events in the negative control. Antibodies and signal detection reagents were obtained from BD Biosciences unless stated otherwise.