Plastics
Standard disposable plasticware was used. Plastics for tissue culture were radiation sterilised by the manufacturers. Further details are given in Appendix I.
Mice
BALB/c (H-2^), CBA (H-2^) and C57BL/6 (H-2t>) female mice were obtained from Biological Services, ICRF Clare Hall Laboratories. They were kept in the animal facilities at the Biological Services Unit, University College London, and used in experiments at 6-12 weeks of age.
Reagents
Unless otherwise indicated, all reagents were obtained from Sigma, BDH, Molecular Probes, Pharmacia, BRL and Boehringer Mannheim and were of the highest grade available (see Appendix II for further details). Storage procedures for certain reagents are shown in Table 2.1.
Cytokines
Cytokines were all purchased in the form of lyophilised protein from suppliers shown in Appendix II. The protein was dissolved in water to lOOng/pl, further diluted in PBS A to
lOng/pl and stored in 50pl aliquots at -80°C.
Radioactive Isotopes
Tritiated thymidine ([^H]TdR, 76.0 Ci/mmol), Na^lCr04 (5mCi/ml) a-p^S]dATP (> 1 OOOCi/mmole) and l^^I (lOOmCi/ml) were all purchased from Amersham.
Na^^Cr0 4 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
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 mg/ml in PBS.
Filtered through 0.2 pm.
Stored protected from light at 4°C for 1-2 weeks maximum.
Transferrin 10 mg/ml in H2O.
Filtered through 0.2 pm. Stored in aliquots at -20°C.
Table 2.1
Storage procedures for certain reagents.
Peptides
Peptides shown in Table 2.2 were synthesised by the Peptide Synthesis Laboratory at ICRF, Lincoln's Inn Fields by standard fmoc chemistry and received in powder form. The quality of the peptide was checked by mass spectrometry and HPLC (by the Peptide Synthesis Laboratory). Peptides which were used to coat dendritic cells in CTL culture or to label RMA-S cells for CTL assays were dissolved at 20mg/ml (~20mM) in DMSO and stored in 20pl aliquots in eppendorf tubes at -80°C. Hen Eggwhite
Lysozyme (HEL) derived peptides were stored in powder form in sealed glass tubes at room temperature in the dark for up to three months. Peptides were freshly dissolved in PBS A at lOx concentration and then diluted in medium for each experiment.
Derivation of peptide MHC which presents peptide
Sequence of peptide
Human Papillomavirus strain 16 (HPVI6) E6 130-137
H-2Kt> GRWTGRCMS
HPV16E7 49-57 H-2Kb, Db RAHYNIVTF
Ovalbumin 257-264 H-2Kb, Db SIINFEKL
HEL 2-16 I-Ed VFGRCELAAAMKRHG
HEL 11-25 I-Ad AMKRHGLDNYRGYSL
HEL 23-32 I-Ad YSLGNWVCAA
HEL 71-85 I-Ad GSRNLCNIPCSALLS
HEL 106-116 I-Ed MNAWVAWRNRC
HEL 20-35 EAk YRGYSLGNWVCAAKFE
HEL 46-61 I-Ak NTDGSTDYGILQINSR
HEL 74-86 I-Ak NLCNIPCSALLSS
Table 2.2
Details of peptides used in these studies. The storage procedure for the peptides is described above.
Media
RPMI: RPMI 1640 supplemented with 2g/litre sodium bicarbonate and 2mM glutamine (ICRF media production).
IMDM: Iscove's modified Dulbecco's medium (Gibco) supplemented with 2mM glutamine, 3.02g/l sodium bicarbonate, 100 units/ml penicillin and streptomycin and 5 pg/ml transferrin.
HL-1: Serum-free medium (BioWhittaker) supplemented with 2mM glutamine and 100 units/ml penicillin and streptomycin. This was kept for a maximum of six months at 4“C.
MEM: Minimal essential medium (ICRF media production).
Sera
Fetal calf serum (FCS, Gibco) was routinely heat inactivated at 56°C for one hour before freezing in aliquots. For in vitro induction of CTL, tested batches of FCS were used directly without heat inactivation.
Buffers
PBSA was obtained from ICRF Clare Hall.
Antibodies
Designation Target molecule Isotype Source Conjugate (if any) Y3 Kb a l -t- a2 domains Mouse IgG2b, K ATGG none
F7D5 Thy-1.2 Mouse IgM Serotec none
Tib 93 I-Ak IgG2b ATGG none
Tib 120 I-A (b, d, q) I-E (d, k)
Rat IgG2b, K
ATGG none
KT3.1.1 CD3 Rat IgG2a ATGG none
Ml/70.15 GDI lb (Mad): macrophages, monocytes, granulo cytes and NK cells
Rat IgG2b Sera-lab none
N418 a-chain of a 150,90 kDa integrin (GDI Ic) on dendritic cells. Not on lymphocytes and macrophages
Hamster mAb Alexandra Livingstone, Imperial Gollege.
biotin
37-51 GD28 Hamster mAb PharMingen biotin
RA3-6B2 GD45-B220: cell surface protein on B lymphocytes and natural killer cells
Rat Ig02a PharMingen biotin
C2 GD71 (Transferrin
receptor)
Rat IgGi, K PharMingen none
In.l GD74 (Invariant
chain)
Rat IgG Anne Sponaas, Berlin
none
GLl GD86 (B7-2) Hamster mAb PharMingen biotin
1D4B GDI07a (LAMP-1) Rat IgG2a, k PharMingen FITG
Table 2.3
Target Ig Host Conjugation Source
Rat IgGib Goat FITC Nordic
Rat IgGib Goat Cy5 Molecular Probes
Rat IgG Rabbit FITC Sigma
Rat Ig Goat FITC Nordic
Mouse Ig (IgA, IgG, IgM)
Goat FITC Sigma
Hamster Ig Goat FITC Nordic
Biotin N/A (Streptavidin) Per CP PharMingen
Table 2.4
Secondary antibodies and streptavidin-Cychrome C used to visualise staining of cells with unconjugated or biotinylated first layer antiodies.
Cell lines
Murine cell lines used in this project are summarised below: those used as targets in CTL assays (Table 2.5), and HEL-determinant specific T cell hybridomas used for antigen processing assays (Table 2.6).
EL4 cells were established in tissue culture from a murine 9:10- dimethyl-benzan- thracene induced thymoma from C57BL/6 mice(Gorer, 1950). RMA-S cells originate from a Rauscher virus-induced C57BL/6 T cell lymphoma RBL-5 which was
mutagenised with ethyl methane sulfonate(Ljunggren and Karre, 1985). PS 15 cells were derived by inducing a murine DBA/2 mastocytoma with methylcholantrene(van Pel,
Cell line Origin MHC class I expression
Growth medium Source
EL4 Murine
thymoma
Kb, Db RPMI/5% FCS ATCC
RMA-S Murine T cell lymphoma Kb, Db RPMI/5% FCS K. Karre, Stockholm. P815 Murine mastocytoma Kd, Dd, Ld RPMI/5% FCS ECACC Table 2.5
Cell lines used as tartgets in CTL assays.
Designation of T cell hybridoma HEL determinant recognised MHC Class II molecule presenting determinant IC5.1 46-61 I-Ak AGIT 74-86 I-Ak Ed2 2-16 I-Ed G28C9 106-116 I-Ed Ad23 23-32 I-Ad Ad71 71-85 I-Ad Table 2.6
Summary of T cell hybridomas used in for antigen processing assays. All hybridomas were from S. Schneider and E. Sercarz, La Jolla Institute for Allergy and Immunology, California, except for IC5.1, from P. Fairchild, Department of Haematology, Cambridge University.
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 which excluded trypan blue were counted.
Cryopreservation and retrieval of cells
Cells for cryopreservation were counted, centrifuged and resuspended in FCS with 10% v/v of DMSO. 5x10^ cells/ml were slowly 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 were retrieved from cryopreservation by quick warming in a 37°C water bath. As soon as the mixture had thawed it was transferred to a T25 flask and fresh medium at 37°C was added drop wise. After overnight incubation cells were split 1:5 into fresh medium and culture was continued at 37°C/5%C02 in a humidified incubator for all cell types unless otherwise indicated.
Immunofluorescence staining of cell surface markers
Cells suspended in PBSA were aliquotted into a 96 well flexible plate (Dynatech microtitre plate) at about 2xl0^/well. They were pelleted by centrifugation at 300 x g for 2 minutes. The supernatant was discarded by inverting the plate and the cells resuspended by agitating on a whirlimixer. The cells were incubated on ice with the appropriate first layer antibody (Table 2.3) at a predetermined optimum dilution for twenty minutes then washed three times with ice cold PBSA. Both directly conjugated and unconjugated antibodies were used in different experiments. For unconjugated or biotinylated antibody a fluorescence conjugated second layer of the appropriate anti species Ig or streptavidin Cy C was used (Table 2.4). When two layer staining was performed some cells were stained only with the second layer as a control for non specific staining. When staining cells for two markers experiments were designed so as
to avoid using second layer antibodies which could bind both first layer antibodies. Stained cells were examined and quantified using a FACScan (Becton Dickinson) with FACScan software.
Enriching dendritic cells from spleen
Dendritic cells were enriched from mouse spleens by taking advantage of two properties of these cells: transient adherence and low buoyant density. Strongly adherent cells (e.g. macrophages) remain stuck to plastic after an overnight incubation whereas dendritic cells do adhere initially, but detach after 2 hours and are non-adherent after an overnight incubation. These non-adherent, low density dendritic cells can subsequently be
separated from other non-adherent, high density cells such as lymphocytes by centrifugation on a layer of metrizamide.
Spleens from young adult mice were teased through a sterile plastic mesh to make a single cell suspension which was then washed once and resuspended at 5x10^ cells/ml in RPMI/10% FCS. 10^ spleen cells were seeded in a T75 flask and incubated
overnight. The following day non-adherent cells were harvested, centrifuged and resuspended at 10^ cells/ml in RPMI/10% FCS. 8 ml of cells were layered on top of 2 ml 14.5% (w/v) metrizamide in RPMI/10% FCS in a universal tube. The cells were centrifuged for 10 minutes at 600 x g (1800 rpm). The low density cells at the interface were collected and washed. These cells were routinely >40% N418+ve dendritic cells (see chapter 3).
Growing dendritic cells from bone marrow precursors
Dendritic cells can be differentiated from precursor cells by incubation with the cytokine GM-CSF. The method employed in this thesis was a variant of that described by Inaba et al (Inaba et al, 1992).Depletion of B lymphocytes from the precursor population by anti-B220 antibody plus complement was found to significantly reduce the resulting yield of dendritic cells at the end of the culture without affecting the purity of the cells, and so was not performed routinely.
Femurs were removed from young adult female mice, placed in MEM/5% FCS, and the marrow within flushed out using a 23G needle (Becton Dickinson) attatched to a 2ml syringe. Cells were vigorously pippeted up and down to create a single cell suspension which was then washed once, counted and resuspended in IMDM/10%FCS/5xl0'^M 2- ME at 3.33x1 O^cells/ml. GM-CSF was added to the cultures at lOng/ml, and the cells were incubated in T25 flasks. After 2-3 days of incubation, media plus non-adherent cells were aspirated off leaving adherent cells and loosely adherent colonies. Fresh IMDM/10%FCS/5xl0"^M 2-ME plus lOng/ml GM-CSF (and sometimes retrovirus) was added. For generating CTL, non-adherent cells were removed after a further 4 days and were generally >70% dendritic cells (see chapter 3). For use in antigen processing assays or endocytosis assays, non-adherent cells and loosely adherent colonies were removed 3 days after the first media change, washed, resuspended at 10^ cells/ml and subcultured overnight in fresh IMDM/10%FCS/5xl0"^M 2-ME plus lOng/ml GM-CSF with or without additional cytokines also added. The yield of non-adherent dendritic cells the next day depended upon which cytokines were present overnight and will be discussed in chapter 4.
Allogeneic mixed lymphocyte proliferation assays
Splenic dendritic cells or bone marrow derived dendritic cells at day 6, 8 and 10 of culture were treated with Mitomycin C for Ihr, washed three times, then incubated at graded doses in triplicate with allogeneic responder cells at 2xl0^/well in 96 well flat- bottomed plates. Responders were non-adherent cells from splenocytes cultured in T75 flasks for 90 minutes (during this time DC and macrophages stick to the plastic). Controls with no dendritic cells and with PH A at Ipg/ml were included in each assay. After 3 days each well was pulsed for 18 hours with lOpl PBSA containing IpCi
[^H]TdR before being harvested onto glass fibre filters using a cell harvester (Skatron). Incorporation of [^H]TdR was assessed by liquid scintillation using the LKB betaplate system.
Induction of cytotoxic T lymphocytes
CTL were generated from mouse spleen cells by priming CTL in vitro from spleens of naive mice using dendritic cells. T cell medium used for in vitro culture of T cells was IMDM/10%FCS/5x10-5m 2-ME.
Using dendritic cells to generate allogeneic CTL in vitro
Splenic or bone marrow derived dendritic cells at 8 x 10^/well were incubated with allogeneic responder cells at 4 x 10^/well (a ratio of 1:50) in 96 well U-bottomed plates in T cell medium for 6 days. Responders were non-adherent splenocytes derived as above for allogeneic mixed lymphocyte proliferation assays.
Using dendritic cells to generate syngeneic CTL in vitro
Syngeneic CTL were generated using peptide pulsed dendritic cells. Dendritic cells were then incubated with syngeneic responder cells (derived as for allogeneic responder cells) at a 1:50 ratio for 6 days as above. 10^ splenic or bone marrow derived dendritic cells RPMI/2% FCS were incubated for 4 hours with 100 pM synthetic peptide in a volume of 2ml then washed 3 times.
CTL assay
Cytotoxic T cell activity was determined in a ^^Cr release assay. The assay uses the release of chromium from pre-labelled target cells to measure lysis of target cells after contact with effector T cells. All assays were carried out in 96 well U-bottom non-tissue culture treated assay plates. The assay medium used was RPMI/5% FCS.
Target cells
All target cells were seeded at mid-log phase growth and >90% viability the night before the assay. RMA-S cells were cultured at 26“C overnight before the assay to stabilise surface MHC Class I. On the day of the assay target cells to be peptide labelled were cultured at 10^ cells in 50pl for 1 hr at 37“C with lOOpM peptide. All target cells
were radioactively labelled with 50 mCi Na^^Cr04 by adding 10 ml of 5 mCi Na^lCr04 for a further hour. Cells were then washed three times, counted and resuspended at SxlO^/ml.
Effector cells
After 6 days of culture of dendritic cells with responders, viable cells were counted, washed and resuspended at 5xlO^/ml in assay medium. Serial 2-fold dilutions in triplicate were then set up in assay plates before addition of target cells.
Replicate wells with 100 ml medium or 100 ml 0.5% (w/v) SDS representing spontaneous and maximum release respectively were prepared on separate plates. Target cells were then added at 5xlO^/well to all wells generating cultures with various effector cell:target cell ratios. Assay plates were then spun briefly once to pellet cells and cultured at 37°C for 4 hr. After this time 100 ml supernatant was harvested from each well and y-radioactivity was counted using a LKB Wallac 1272 Clinigamma y- counter linked to an Olivetti PCS 286 computer. The "% specific release" was calculated as:
(experimental release-spontaneous release") ^ \qq%.
(maximum release - spontaneous release)
MHC Class I peptide binding assay
This assay measures the ability of peptides to stabilise empty murine MHC class I on the surface of RMA-S cells at 37°C. These are RMA cells with a defect in surface class I expression at 37“C due to the lack of a functional TAP 2 protein. Prior to the assay, RMA-S cells were cultured at 26“C overnight, this induces up-regulation of expression of empty class I molecules(Ljunggren et al, 1990; Schumacher et al, 1990). The assay medium was RPMI/2% FCS which had been heated for 10 minutes at 100°C in a waterbath to inactivate proteases. Serial dilutions of peptides were prepared in assay medium in 96 well, U-bottom, non-tissue culture treated plates. 10^ temperature-
induced RMA-S cells were added to each well, and to wells containing no peptide (negative control), and some RMA-S cells were kept on ice as positive controls. The plates were incubated at 37°C for 2hr to allow the peptides to stabilise MHC Class I. After this these cells plus cells kept on ice were washed once with PBS A/2%
FCS/0.01% (w/v) NaNg, stained with anti-murine class I Y3 antibody and analysed by FACS analysis as described below. Half maximal binding for an individual peptide was determined as the concentration of peptide needed to obtain 50% of the maximum mean FLl signal obtained for the peptide when using the mean FLl obtained with RMA-S cells incubated without any peptide (negative control cells) as baseline.
Antigen processing assays
Dendritic cells from female CBA or BALB/c mice were treated in various ways (described in detail in chapter 4) and cultured at 10“^ cells/well with HEL-determinant specific hybridoma cells at 5 x 10^ cells/well with whole HEL or HEL peptide at graded doses, in 96-well U-bottomed plates. The medium used was
RPMI/5%FCS/5x 10'^M 2-ME. After 24 hours supernatants were removed and IL-2 levels were assayed by proliferation of the IL-2 dependent cell line CTLL at 5x10^ CTLL cells/well in a 96-well U-bottomed plate measured by [^H]TdR incorporation over the last 18 hours of a 36 hour culture. Controls with serial dilutions of recombinant IL-2 were always included. Neither supernatants of dendritic cells alone nor of any hybridoma elicited CTLL proliferation of more than 2000cpm. Results are expressed as the mean of triplicates, with all values falling within 10% of the mean.
Immunisation of mice with dendritic cells
Dendritic cells from female CBA or BALB/c mice were cultured with whole HEL at 50pg/ml for 6 hours, washed three times, resuspended at 10^ cells/ml in PBSA and 5 x
10"^ cells/foot were injected into opposite hind footpads of 6 female CBA or BALB/c mice. 5 days later draining popliteal and inguinal lymph nodes from either side of the mice were pooled into MEM. Nodes were put though a sterile plastic mesh to make a single cell suspension and lymphocytes were then cultured in HL-1 serum free medium
at 5 X 10^ cells/well in 96-well U-bottomed plates in triplicate with HEL, ovalbumin or HEL peptides at 60 pg/ml for 4 days. Proliferation was measured by [^H]TdR
incorporation over the last 18 hours of culture.
Immunisation of mice with protein
HEL (lOOpg/mouse) plus or minus IL-6 (5ng/mouse) in PBSA was emulsified at 1:1 v/v ratio in lOOpl IFA, then injected at the base of the tail into 5 female CBA or
BALB/c mice. 10 days later harvested spleens were put through a sterile plastic mesh to make a single cell suspension and splenocytes were cultured at 10^ cells/well for 5 days in HL-1 medium as above. Background proliferation values without added antigen (-2000 cpm) were subtracted.
Antigen presenting cell separation
HEL plus or minus IL-6 as above was emulsified in IFA as above and injected into opposing rear footpads of 10 female BALB/c mice. In a separate experiment lOOpg/foot ovalbumin plus 5ng/ml IL-6 in IFA were immunised into the rear footpads of 10 female BALB/c mice. The total volume injeceted per foot was less than 40pl. 5 days after immunisations draining nodes were harvested and a single cell suspension made. Cells were then incubated with saturating amounts of anti-Thy 1.2 antibody in RPMI/5%FCS on ice for 30 min, then washed twice with ice-cold medium and resuspended in serum-