In this chapter general materials and m ethods will be described. More specific points on m ethodology relevent to each chapter can be found at the beginning of each of the result chapters.
Plastics
Standard disposable plasticware m anufactured by Falcon, UK, w as used that were supplied by ICRF. Plastics for tissue culture were radiation sterilised by the manufacturers.
Mice
C57BL/6 (H-2b), BALB/c (H-2<1) and CBA (H-2k) fem ale mice w ere obtained from Biological Services, Imperial Cancer Research Fund (ICRF) Clare H all Laboratories. They were kept in the anim al facilities at the Biological Services Unit, U niversity College London, and used in experim ents at 6-12 weeks of age.
Reagents
Unless otherwise indicated, all reagents w ere obtained from Sigma or BDH and w ere of the highest grade available. Storage procedures for certain reagents are show n in Table 2.1.
Radioactive Isotopes
Tritiated thym idine (PH]TdR, 76.0 C i/m m ol), N a^^Cr04 (5m C i/m l), y-
purchased from Amersham. Na^^Cr04 was used for up to two weeks after the delivery date, and then allowed to decay.
Reagent Procedure used to prepare
and store stock solutions
Cytokines lyophilised protein was
dissolved in water to lOOng/pi stored in 25pl aliquots at -80°C
p-mercaptoethanol (2-ME) 5 X 10'2 M in IMDM medium.
Filtered through 0.2 pm. Stored in aliquots at -20°C.
Mitomycin C 1 m g/m l in PBS.
Filtered through 0.2 pm. Stored protected from light at 4°C for 1-2 weeks maximum.
Table 2.1: Storage procedures for certain reagents.
Peptides and Proteins
SV-9 peptide shown in Table 2.1 was synthesised by the Peptide Synthesis Laboratory at ICRF, Lincoln's Inn Fields and received in pow der form. The quality of the peptide was checked by mass spectrometry and HPLC (by the Peptide Synthesis Laboratory). Tetanus toxoid Fragment-c-7 (Fr-c-7) peptide (King, Spellerberg et al. 1998) was a kind gift from Dr. C.A. King of the Molecular Immunology Group, Southampton University Hospital, UK. To coat target cells for CTL assays, these peptides were dissolved at 20m g/m l (~20mM) in DMSO and stored in 20pl aliquots in eppendorf tubes at -80°C. Peptides were freshly dissolved in PBSA at lOx concentration and then diluted in medium for each experiment.
Human carcinoembryonic antigen (hCEA) was initially extracted from a hum an colorectal metastasis using a perchloric acid extraction technique followed by a fast performance liquid chromatography step. This procedure proved to yield hCEA that was contaminated with unacceptable levels of EPS for our purposes. Subsequently, a com m ercial hCEA (Calbiochem - Novabiochem (UK) Ltd., Cat. No. 219369) was used that was purified from a established human cell line and was delivered in a lyophilised solid form.
Derivation of peptide MHC which presents peptide
Sequence of peptide
Tetanus Toxoid Fr-c-7 H-2Kb SNWYFNHL
Sindai Virus 9 nucleoprotein 324-332
H-2Kb&Db FAPGNYPAL
Table 2.2: Details of peptides used in these studies.
Media
M edia used fo r mammalian cell cultures
RPMI: RPMl 1640 supplemented with 2g/litre sodium bicarbonate and 2mM L-glutamine (ICRF media production).
IMDM: Iscove's modified Dulbecco's medium (Gibco) supplem ented with 2mM L-glutamine, 3.02g/l sodium bicarbonate, 100 u n its/m l penicillin and streptomycin and 5 p g /m l transferrin.
DMEM: Dulbecco's modified Eagles's m edium (Gibco) supplem ented w ith 4mM L-glutamine, 1.5 g/1 sodium bicarbonate, 100 u n its/m l penicillin and streptomycin.
MEM: Minimal essential medium (ICRF m edia production).
M edia used fo r bacterial cell cultures Luria-Bertani (LB)
Bacto-tryptone lOg
Bacto-yeast extract 5g
NaCl lOg
Dissolved in 1000ml d H 2 0 and pH adjusted to 7.5 w ith 5M NaOH. The m edium was then autoclaved.
2TY
Bactro-tryptane 16g
Yeast Extract lOg
Sodium Chloride 5g
dissolved in 1000ml dH2 0 and autoclaved. LB Agar
Bacto-agar 15g added to 1000ml of LB m edium and re-autoclaved. For plates containing ampicllin, 50pg/m l Ampicillin was added prior to pouring.
Sera
Fetal calf serum (PCS, Gibco) was routinely heat inactivated at 56 “C for one hour before freezing in aliquots.
Buffers
Phosphate buffered saline Dulbecco's A (PBSA, 137 mM NaCl, 3.3 mM KOH, 1.7 mM anhydrous KH2PO4, lOmM anhydrous N a2 H P 0 4 adjusted to pH7.4 w ith HCl) was obtained from ICRF Clare Hall. The recipes for all the other buffers are given where they are m entioned in the text.
Antibodies
Designation Target molecule Isotype Source Conjugate
(if any)
1H12 hCEA Mouse IgGi ATCC none
A5B7 hCEA Mouse IgGi ATCC none
EA77 hCEA Mouse IgGia ATCC none
C6G9 hCEA Mouse IgGi Sigma none
L3T4 Mouse CD4 Rat IgG2a,K Pharmingen Biotin
Ly-2 Mouse CDSa Rat IgG2a,K Pharmingen Biotin
L3T4 Mouse CD4 Rat IgG2a,K Pharmingen EITC
Ly-2 Mouse CDSa Rat IgG2a,K Pharmingen FITC
Ly-2 Mouse CDSa Rat IgG2a,K Pharmingen PE
A85-1 Mouse IgGi Rat IgGpK Pharmingen HRP
R12-3 Mouse IgCzb Rat IgG2a,K Pharmingen Biotin
R19-15 Mouse IgC2a and
IgG2c
Rat IgGi Pharmingen HRP
Red-T/ G297-289 Mouse IL-12 p35 subunit of p70 Hamster IgG /Rat IgG2a Pharmingen none C17.8 Mouse IL-12 p40 subunit of p70
Rat IgG2a Pharmingen Biotin
Y3 Kb
a l + a2 domains
Mouse IgG2b, k
ATCC none
Tib 93 I-Ak IgG2b ATCC none
Tib 120 I-A (b, d, q) I-E (d/ k) Rat IgG2b, k ATCC none 70
M l/70.15 C D l l b ( M a d ) : macrophages, monocytes,
granulo-cytes and NK cells
Rat IgG2b Sera-Iab none
N418 a-chain of a 150,90
k D a i n t e g r i n ( C D11 c ) o n dendritic cells. Not on ly m p h o c y te s and macrophages Ham ster mAh Alexandra Livingstone, Imperial College. biotin
RA3-6B2 CD45-B220 Rat IgG2a Pharm ingen biotin
KT3.1.1 CD3 Rat IgG2a ATCC none
16-lOAl CD80 (B7-1) Ham ster
mAh
Pharm ingen biotin
GLl CD86 (B7-2) Ham ster
mAh
Pharm ingen biotin
NLS-G Mouse NF-kB p50 Goat
polyclonal IgG
Santa Cruz none
K-27 Mouse NF-kB p52 Rabbit
polyclonal IgG
Santa Cruz none
C-20-G Mouse NF-kB p65 Rabbit
polyclonal IgG
Santa Cruz none
T able 2.3: Prim ary (1st layer) antibodies used in cell staining and w estern blot analysis. Target molecules are all murine.
Target Ig Host Conjugation Source
Rat IgGzb Goat FITC Nordic
Rat IgG Rabbit FITC Sigma
Rat Ig Goat FITC Nordic
Mouse Ig (IgA, IgG, IgM)
Rabbit HRP Dako
Hamster Ig Goat FITC Nordic
Biotin N /A
(streptavidin)
HRP Zymed
Biotin N /A
(streptavidin)
Mini-Macs beads Miltenyi Biotech
Biotin N /A (streptavidin) FITC Pharmingen Biotin N /A (streptavidin) Cy5 Pharmingen
Table 2.4: Secondary antibodies and Streptavidin-Cychrome C used in ELISA assays, in cell seperation experiments or to visualise staining of cells with unconjugated or biotinylated first layer antibodies.
Cell lines
The cell lines used in this project are summarised below (Table 2.5).
Cell line Origin MHC
expression
Growth medium Source
EL4 Murine
thymoma Kb Db
RPMI/5% ECS ATCC
RMA Murine T cell
lymphoma Kb, Db
RPMI/5% ECS K. K à r r e ,
Stockholm.
P815 Murine
mastocytoma Kd, Dd, Ld
RPMI/5% ECS ECACC
CMT-93 Murine
colorectal carcinoma
H-2b RPMI/5%ECS ICRE
COS-7 African green
monkey renal carcinoma DMEM/5%ECS ATCC CBl Immortalised murine dendritic cell line H-2d I-A & I-Ed
RPMI/5%ECS P.Paglia,
Italy
Table 2.5: Cell lines used for targets in CTL assays and for transfection of hCEA.
Cell counting
lOpl of cell suspension was diluted 1:1 with trypan blue solution (0.2% w /v in PBSA with 3mM NaNg). The cells were then viewed under phase and counted on an improved Neubauer counting chamber using a light microscope. Viable cells that excluded trypan blue were counted.
C ryopreservation and retrieval o f cells
Cells for cryopreservation were counted, centrifuged and resuspended in FCS w ith 10% v / v of DMSO. 5x10^ c e lls /m l w ere slow ly frozen in 1ml cryotubes (Nunc) in an expanded polystyrene block at -80°C for 24-48 hours and then transferred to the vapour phase of liquid nitrogen.
Cells w ere retrieved from cryopreservation by quick w arm ing in a 37°C w ater bath. As soon as the m ixture had thaw ed it was transferred to a T25 flask and fresh m edium at 37°C was added dropw ise, centrifuged and resuspended in fresh m edium . After overnight incubation cells w ere split 1:5 into fresh m edium and culture w as continued at 37°C/5%C02 in a hum idified incubator for all cell types.
Transfection of Mammalian cells
Cells in suspension w ere transfected by electroporation and adherent cells by lipofection.
Electroporation
The cells used for electroporation were split 1:2 at m id-log phase grow th and >90% viability for 2 days prior to electroporation. For each electroporation 5x10^ cells were used. After two washes in cold PBS the cells w ere resuspended in 600pl of IMDM to which 25pg of plasm id DNA or d H 2 0 as control and 50pl of Salmon sperm DNA (at lO m g/m l and freshly boiled) w ere ad d ed . The m ixture w as then transfered to cold 4 m m electroporation cuvettes (BioRad, UK) and placed on ice for 5 m inutes and then electroporated in a BioRad electroporation apparatus as follows;
RMA 200V 960pF P815 800V 25pF
EL4 450V 125^F
After electroporation the cells were im m ediately placed on ice for a further 5 m inutes and then the volume w as m ade u p to 10 m l w ith IM DM /10% PCS. Electroporated cells were transfered to 5 wells of a 24 well tissue culture plates (Falcon, UK) and incubated at 37°C/5% CO2 in a hum idified incubator for 48 hours. The culture m edium w as then changed to R P M I/7.5% ECS and the appropriate selection antibiotic was added. The concentration of antibiotic was d eterm in ed for each cell line prior to electroporation by determ ining the m inim um antibiotic required to kill all the cells th at w ere seeded at 4x10^ c ells/m l, in 3-4 days. Using the lim ited dilution m ethod single cell colonies w ere grow n in U-bottom 96 well tissue culture plates (Falcon, UK).
Lipofection
A dherent cells were split in T-25 tissue culture flasks (Falcon, UK) and transfered to six well tissue culture plates (Falco, UK) for a few days before lipofection to ensure that they were betw een 40-60% confluent on the the day of transfection. The m edium was taken off and the cells w ere w ashed twice w ith serum free m edium (SFM) and replaced by 3 ml of . Plasm id DNA at 30pg in 300pl of SFM or SFM alone and 60pl of lipofectamine reagent (GIBCO BRL, UK) in 300pl of SFM were ad d ed together and left at room tem p ratu re for 15 m inutes. The volumes of the transfection mixture or control w ere m ade u p to 3 m l w ith SFM and to each well 1 ml w as added. The flasks w ere placed in a hum idified incubator at 37°C/5% CO2 overnight. The m edium w as replaced the next day w ith the m edium supplem ented w ith FCS. The a p p ro p riate selection m edium was used as soon as there was a sign of cell growth. A few days after all the controls were dead, single cell colonies could be detected that w ere rem oved using sterile forceps and filter paper and placed along w ith the
filter p ap er in 24 well tissue culture plates (Falcon, UK) initially in 250pl of m edium that w as raised gradually to 1 ml as the cells stared growing.
Immunofluorescence staining of cell surface markers
Cells suspended in PBSA were aliquotted into a 96 well flexible plate (D ynatech m icrotitre plate) at about 2x10^/w e ll. They w ere p elleted by centrifugation at 300 x g for 2 m inutes. The su p ern atan t w as discarded by inverting the plate and the cells resuspended by agitating on a whirlimixer. The cells were incubated on ice w ith the appropriate first layer antibody (Table 2.3) at a predeterm ined optim um dilution for tw enty m inutes then w ashed three tim es w ith ice cold PBSA. Both directly conjugated an d u n co n ju g ated a n tib o d ies w ere u sed in different ex p erim en ts. For u n co n ju g a te d or biotinylated antibody a fluorescence conjugated second layer of the appropriate anti-species Ig or streptavidin FITC w as used (Table 2.4). W hen tw o layer staining w as perform ed some cells were stained only w ith the second layer as a control for non-specific staining. W hen stain in g cells for tw o m arkers experiments were designed so as to avoid using second layer antibodies which could bin d bo th first layer antibodies. Stained cells w ere exam ined and quantified using a FACScan (Becton Dickinson) w ith FACScan software.
Intracellular staining and confocal microscopy
All staining steps were carried out on perm eabilised cells in the dark at room tem perature. Cells were fixed and perm eabilised by adding to each well 50pl of PermeaFix (Ortho) diluted 1:2 in PBS . Plates were then agitated in the d ark for 40 m in at room tem perature. Cells w ere then w ash ed in RPMI supplem ented w ith 10% FCS, twice. 1st layer biotinylated oligonucleotides
w ere added, and cells were agitated for 30min, w ashed twice, and th en 2nd layer antibody w as added and left for 30 m in and w ashed as above. Cells were then w ashed twice, fixed in PBSA/1% parafom aldehyde, w ashed twice m ore and then resuspended in 50pl PBSA. Controls of second layers w ith o u t first layers, and of staining one target molecule only w ere also included.
M ounting cells onto slides
50pl cell suspensions were pipetted onto poly-L-lysine coated 6mm diam eter wells of 8-well glass slides (Roboz Surgical Instrum ents) and left at 4°C for 30min to allow the cells to adhere. Excess PBSA was then rem oved from the w ells, and a drop of m ounting m edium (20% glycerol plus anti-fade reagent) w as added before a cover slip w as low ered and secured w ith Jet Set Red nail varnish (Rimmel). Slides were stored at 4°C.
Confocal microscopy
M ounted cells w ere viewed th ro u g h an inverted Zeiss A xiovert 100 m icroscope in conjunction w ith a Bio-Rad MRC 1024 Laser Scanning Confocal Im aging System. This uses a krypton-argon laser ru n by LaserSharp softw are in an O S /2 W arp C onnect operating system w hich acquires digital im ages. R epresentative im ages w ere then processed u sin g A dobe P hotoshop and M icrosoft Pow erpoint software, and printed on Epson photographic quality p ap er by an Epson Stylus colour printer.
Enzyme Linked Immunosorbent Assay (ELISA)
Im m unoglobulins directed against hCEA from mice im m unised w ith hCEA vectors or hCEA protein were detected by ELISA. At a desired tim e point tail bleeds w ere perform ed under anaesthesia. Blood was allow ed to coagulate
in eppendorf tubes at room tem perature for 4 hours, then tubes w ere spun at 13,000rpm for 4 minutes. Sera was rem oved into fresh eppendorfs and stored at -20°C before use in ELISA.
ELISA plates (DynaTech) w ere coated w ith 5 0 p l/w e ll of 2 p g /m l hCEA (C albiochem -N ovabiochem Corp., UK) in coating buffer (8.4 g / I sodium bicarbonate at pH 8.2 in millipore dH 2 0 ) at 4°C overnight. Next day all wells were w ashed (using washing buffer, PBS-A w ith 0.05% Tween 20) three times and then blocked w ith 150pl of PBS-A/1% Bovine serum albumin(BSA) at room tem p eratu re for 1 hour. Wells were w ashed as above and 50pl of sera or standard antibodies against hCEA were added in serial dilutions and incubated at room tem perature for 2 hr. Wells w ere w ashed as above and then 50pl of 1:400 H orseradish peroxidase (HRP)-conjugated rabbit anti m ouse Ig (Dako) or 1:500 HRP-conjugated anti-mouse isotypes (IgGi or IgC2c from Pharm ingen ) added for Ih r at room tem perature. After w ashing, 50pl of substrate solution (Dako TMB One-Step substrate solution) was ad d ed to each well an d plates w ere incubated in the dark at room tem perature until the colour developed. 25pl stop solution (10% sulphuric acid) was then ad d ed to each well and the OD450 of each well was read using a Dynatech MR700 m icroplate reader.
Sandwich ELISA
Sandw ich ELISA was used to detect cytokine levels in su p ern atan t of bone m arrow derived dendritic cells treated w ith oligonucleotides. The m ethod used for detection of interleukin-12 (IL-12) ELISA will be described below as only the results of this assay are reported in this thesis. This assay had a m inim um IL-12 detection level of 16 n g /m l.
ELISA plates (DynaTech) w ere coated w ith lOO pl/well of lO p g /m l IL-12 capture antibody cocktail at 4°C overnight. The capture antibody cocktail w as a
com m ercially p rep ared m ixture of two antibodies. Red T and G297-289 (Pharm ingen, UK) that bind the mouse IL-12 p35 subunit of the IL-12 p70 heterodim er and the coating buffer used was O.IM N a2H P 04 at pH9. N ext day all wells were washed (using washing buffer, PBS-A w ith 0.05% Tween 20) four times and then blocked with 200pl of PBS-A/1%BSA at room tem perature for 1 hour. Wells were w ashed three times and lOOpl of the sam ples or stan d ard IL- 12 (recom binant mouse IL-12, Pharmingen, UK, a range of 2.3 to 1667ng/m l) w ere added serially diluted in blocking buffer and incubated at 4°C overnight. W ells were w ashed four times and then lOOpl of 1:500 of C17.8 in blocking buffer (biotin-conjugated rat anti mouse IL-12 monoclonal antibody th at binds the p40 subunit of IL-12 p70, Pharm ingen, UK) w as ad d ed for Ih r at room tem perature. The detection aantibody was then w ashed five times and lOOpI of HRP-conjugated streptavidin (ZYMED, USA) diluted 1:4000 in blocking buffer w as ad d ed for 30 m inutes at room tem prature. After six w ashes, lOOpl of substrate solution (Dako TMB One-Step substrate solution) w as added to each well and plates were incubated in the dark at room tem perature until the colour developed. 50pl stop solution (10% sulphuric acid) was then added to each well