B cell purification and induction of apoptosis
PBMC were washed three times with PBS, absorbed on nylon wool columns (Biotest,
Breiech, Germany), and incubated at 37C for 1h. Unabsorbed cells were removed by
extensive washing with warm RPMI and B cells were eluted from the column (14). Effluent B
cells were collected purity was determined by flow cytometry (91.31.4 % meanSEM).
Induction of apoptosis was performed according to the modified method (15). Briefly 6 x 106
tumour B and normal cells were subjected to 5Gy  irradiation (Alcyon, CGR, Paris, France).
Irradiated cells were suspended in medium and incubated at 37 C and 5% CO2 for 24h.
T cell purification
PBMC were washed three times with PBS and passed through a nylon wool column
(Biotest). The effluent cells were incubated with anti-CD19, CD14, and anti CD56
MidiMACS beads (Miltenyi Biotec, Bergisch Gladbach, Germany) according to the
manufacturer’s instructions and passed through MidiMACS column (Miltenyi Biotec). The
purity was checked by flow cytometry (891.9% meanSEM). Cells were resuspended in
RPMI 1640 medium (Gibco, BRL, UK) supplemented with 10% heat-inactivated pooled
human AB+ serum, 2 mM L-glutamine, 100 IU/ml penicillin and 100g/ml streptomycin
(referred to as complete medium).
Enzyme- Linked ImmunoSpot Assay (ELISPOT)
Identification of IFN- secreting T cells was performed as described earlier (17). Apo-DC,
DC-lysate, and DC-RNA (2x104) were added to 2x105 autologous T cells and incubated
overnight at 37 C. Ninety six well flat-bottomed nitrocellulose micro titer plates (Millipore
AB, Stockholm, Sweden) were coated with anti-human IFN- MAb clone 1-D1K;
MABTECH, Sweden) at 4C over night. The antibody-coated plates were then washed with
sterile PBS and pre-incubated cells were added to the wells and incubated at 37 C for 24h.
Biotinylated anti-IFN- avidin-conjugated alkaline phosphatase and BCIP® (Sigma
Diagnostic, St. Louis, MO) were used for developing the color reaction. Spots were counted
using an automatic ELISPOT reader (Axioplan2 Microscope reader, Carl Zeiss, Jena,
Germany). Similar experiments were also performed using normal DC and B cells from
healthy volunteers.
RNA extraction and cDNA synthesis
T-cells were stimulated in 12-well tissue culture plates (Corning Incorporated 3513, Corning,
NY). 2106 T cells and 2105 Apo-DC, DC-lysate, or DC-RNA were cocultured for 18 h in 2
ml RPMI 1640 complete medium. T cells alone and T cells + unpulsed DC were used as
6
controls. Total RNA was extracted from 2x10 T cells, using RNAzol™ B (TEL-TEST Inc,
Friendswood, Texas) using guanidine thiocyanate phenol-chloroform extraction method,
according to the manufacturer’s recommendation. RNA was denatured at 65 °C for 5 min and
immediately chilled on ice. First-strand cDNA synthesis was performed in a 20 l reaction
mixture containing 0.5 g RNA in 10 l volume, 4 l 5x buffer (GIBCOBRL, Gaithersburg,
MD), 1.5 l dithiothreitol (DTT, 100 mM), 2 l dNTPs (5 mM each, Amersham Biosciences),
1.0 l random hexamer primers (100 pmol/l, Amersham Biosciences), 0.5 l dH2O, 1.0 l
M-MLV reverse transcriptase (200 U/l, GIBCOBRL, Gaithersburg, MD USA). The reaction
mixture was incubated at 42 °C for 45 min followed by 5 min at 70 °C to inactivate reverse
transcriptase and then stored at –20 °C.
Real Time quantitative PCR;
The cytokine gene expressions were quantified using ABI prism 7700 Sequence Detection
system (PE Applied Biosystems, Foster city, CA, USA) as previously described (18).
Primers were designed to span exon junction to prevent amplification of any possible
contaminating genomic DNA (Table 2). Probes were labeled with FAM (6-carboxyfluoresciein) and TAMRA (6-carboxy-tetramethyl-rodamine). Fluorescence signals were
generated during each PCR cycle via 5`-3`exonuclease activity of Ampli Tag Gold DNA
polymerase (18).The relative quantitative expression of different cytokine genes in control
and subjects cells were determined using the arithmetic equation 2-CT according to PerkinElmer instruction manual. Equal amounts of total RNA were used from all samples to
synthesize cDNA using a large volume master mix and equal volumes of cDNA were
subsequently used in all assays. The amount of target gene was normalized to an
endogenous reference gene (-actin) at each stage. The calculation of CT involves
subtraction of  Threshold of Cycle (CT) in control cells from that in activated cells for
each time point. The relative increase of each cytokine was equal to 2-(CT
subjects - CT
. CT for control or subject was calculated by subtraction of the endogenous control
controls)
CT from the target cytokine CT. Accordingly the stimulation index (SI) by RT-QPCR was
defined by dividing the relative increase in different cytokine gene expressions in
stimulated samples, by those of unstimulated samples.
T cell proliferation assay
To assess the capability of Apo-DC, DC-lysate, and DC-RNA, to induce an autologous, T cell
proliferative response, 2x104 of each of Apo-DC, DC lysate, or DC-RNA were added to
2x105 autologous T cells in a 96-well U-bottom plate in triplicates and incubated at 37 C for
5 days. T cells alone and with unpulsed DC were used as controls to determine baseline
proliferation. 3H-thymidine (Amersham Pharmacia biotech) was then added to each well at a
concentration of 1 Ci for the final 18 h of culture. The plates were harvested and the
incorporated 3H thymidine was quantitated using a beta scintillation counter (Microbeta
1450, Wallac, Turku, Finland). Proliferative responses of autologous T cells from normal, age
matched donors induced by Apo-DC, DC-lysate and DC-RNA, using autologous, normal B
cells as the antigen source, were also examined (n=3).
Sequence of primers and probes for cytokines and ß-actin used in real-time PCR
Target sequence Primer
Sequence (5’3’)
Accession no. Position
-actin
Sense
CGACAGGATGCAGAAGGAGA
Probe
(X)AAGATCAAGATCATTGCTCCTCCTGAG(Y)
Antisense CGTCATACTCCTGCTTGCTG
NM-001101
NM-001101
NM-001101
1003-1022
1049-1075
1144-1163
IFN-
Sense
TCTGCATCGTTTTGGGTTCT
Probe
(X)TGACCAGAGCATCCAAAAGAGTGTG(Y)
Antisense GCAGGCAGGACAACCATTACT
X13274
X13274
X13274
143-162
363-387
605-625
GM-CSF
Sense
ACACAGCCCTGGGAGCAT
Probe
Antisense (X)ACAGCACTGCCCTCCAACCCC(Y)
CTGGCCGGTCTCACTCCT
M11734
M11734
M11734
94-111
321-341
443-459
TNF-
Sense
TGTTGTAGCAAACCCTCAAGC
Probe
(X)ACACCATCAGCCGCATCGC(Y)
Antisense AGTCGGTCACCCTTCTCCA
AF043342
AF043342
AF043342
45-65
233-251
377-395
IL-2
Sense
GAATGGAATTAATAATTACAAGAATC
Probe
(X)ACATGCCCAAGAAGGCCACAGAACTG(Y)
Antisense ATGTTGTTTCAGATCCCTTTAGTTCCCAGA
U25676
U25676
U25676
191-216
250-275
395-423
IL-4
Sense
TCTGTTCTTCCTGCTAGCA
Probe
Antisense (X)TGCTGCCTCCAAGAACACAACTGA(Y)
CCGTTTCAGGAATCGGATCA
M13982
M13982
M13982
93-111
234-257
371-390
IL-10
Sense
GCTGGAGGACTTTAAGGGTTAC
Probe
(X)AGAACCAAGACCCAGACATCAAGGC(Y)
Antisense ACAGGGAAGAAATCGATGACAG
M57627
M57627
M57627
240-261
326-350
405-426
Perforin
Sense
CCAGAAGACCCACCAGGACCAGT
NM-005041
Probe
(X)TGCCGCTTCTACAGTTTCCATGTGGTACAC(Y) NM-005041
Antisense GGGTGGAGGCGTTGAAGTGGTG
NM-005041
X:TAMRA
Y:FAM
477-499
526-555
604-625