Chemicals
Acrylamide Bisacrylamide Solution (40% w/v 19:1) - Northumbria Biologicals Ltd Agarose (electrophoresis grade) low and high melting point, ultrapure - BRL(Bio-rad labs) Ammonium Acetate (7.5M stock) - Sigma
Ammonium Persulphate (10%) - BRL Ampicillin (50mg/ml stock) - Sigma Bromophenol blue - Sigma
BSA (bovine serum albumin) fraction V - Sigma Chloroform - BDH
Deoxyribonucleoside triphosphates (4mM stock) - Pharmacia Dextran Sulphate - Sigma
Dithiothreol (lOmM stock) - Sigma
DNA Salmon sperm (sodium salt type III), lOmg/ml stock - Sigma DNA size markers - Lamda Hindlll DNA - Biolabs
- 0x174 Haelll DNA - Biolabs Ethanol (100%) - BDH
Ethidium Bromide (lOmg/ml stock) - Sigma
Ethylenediaminetetraacetic acid (EDTA) 0.5M pHS.O - BDH FITC - Becton Dickinson
Formaldehyde (40%) stock - BDH Hydrochloric acid (concentrated) - BDH Isopropanol - BDH
Magnesium Chloride (IM stock) - BDH Methanol - BDH
Penicillin/Streptomycin (10,000U/ml) - Sigma
Phenol (equilibriated with Tris.Cl O.IM pH 8.0 and 8-hydroxyquiniline 1%) - Rathborn Chemicals Ltd.
Phycoerythrin coupled streptavidin - Becton-dickinson Potassium acetate (5mM stock) - Sigma
Potassium cloride (IM stock) - Sigma
Radiochemicals -[y-^^P] dCTP (3000Ci/mMol) - Amersham -[a-3^S] dATP (1500Ci/mMol) - Amersham - ^H - thymidine - Amersham
Sephadex G50 DNA grade (medium) - Pharmacia Sodium chloride (5M stock) - Sigma
Sodium citrate - Sigma
Sodium hydroxide (lOM stock) - BDH Spermidine (IM stock) - BDH
TEMED ultra-pure - BRL
Trisbase (IM pH7.4 and pH8.0) - BRL Urea ultra-pure - BRL
Whatman 3mm filterpaper - Whatman
Enzymes
Alkaline phosphatase (calf intestine): DNase (RNase free):
RNase: RNA Guard: Boehringer Mannheim Pharmacia Pharmacia Pharmacia Lysozyme (grade 1 from chicken egg white) Sigma
Proteinase K: Boehringer Mannheim
Restriction Enzymes
EcoRI (25,000 U/ml) Pharmacia BamHI (20,000 U/ml) Pharmacia PstI (15,000 U/ml) Pharmacia S ad ( 15,000 U/ml) Pharmacia Xbal (20,000 U/ml) Pharmacia Nrul (10,000 U/ml) Pharmacia
Polymerases
Taq DNA polymerase AMV reverse transcriptase:
Klenow fragment with Multiprime kit:
Gibco BRL Pharmacia
Amersham
Buffers
All solutions for DNA preparation and analysis were prepared in double distilled water and autoclaved and filtered.
Sephadex G50 (medium) Buffer: 150mM NaCl, lOmM EDTA pH 8.0. 0.1% SDS, 50mM Tris.Cl pH 7.4
20X SSC pH 7.0: 3M NaCl, 3M Na3citrate.2H2Ü, pH to 7.0 with IM HCl
TE pH 7.4/8.0: lOmM Tris.Cl, pH 7.4/8.0, ImM EDTA pH 8.0, Tris.Cl pH 7.4/8.0
Restriction endonuclease buffers: lOx low, medium or high: (NaCl concentration depends on endonuclease), lOOmM Tris.Cl pH 7.5,
lOOmM MgCl2, lOmM DDT, 0, 0.5 or l.OM NaCl
Gel sample loading buffer lOx: 20% Ficoll 400, 0.1 M EDTA pH 8.0,1.0% SDS, 0.25% Bromophenol Blue
Denhart's solution: lOOx (filtered and stored at -20^C in 50ml aliquots): 2% (w/v) Ficoll 400; 2% polyvinylpyrrolidone (w/v), 2% BSA (w/v) (Pentax Fraction)
Denaturing buffer: 0.5M NaOH, 1.5M NaCl
Neutralising buffer: 1.5M NaCl, IM Tris.Cl pHS.O
Prehybridisation solution 5x: lOx Denhardt's solution, 3x SSC, 0.5% SDS, 50mg/ml sonicated salmon sperm DNA, 0.1 mg/ml herring sperm DNA
Hybridisation solution: Prehybridisation solution plus 5% dextran sulphate
Hybond N wash buffer 1 IxSSC, 0.1%SDS
Hybond N wash buffer 2 O.lxSSC, 0.1%SDS
Cultured Cell lysis buffer: 30mM Tris.Cl pH 7.5, lOmM MgAc, 1% Nonidet P-40
Extraction buffer: 2.42g Tris.Cl, 6.72g Na2EDTA.2H2 0, 2.98g KCL, dissolve in distilled water, pH to 7.5 and make up to 2 0 0ml total
Media
L-agar was obtained from the media department at the CSC. All reagents were obtained from Difco. Solutions were autoclaved for 25 minutes.
Solution A: 12g Bactotryptone, 24g yeast extract, 5ml glycerol made up to 900ml with double distilled water and autoclaved Solution B: 62.5g K2HPO4, 19g KH2PO4 made up to 500ml with double distilled water and autoclaved
Antibiotics: Ampicillin 50|Lig/ml
All media and antibiotics for cultured cells, except G418 and HLl media, were made up by the CSC media department with reagents obtained from Gibco.
Antibiotics: Penicillin/streptomycin 50,000 U/ml each
CRPMI (complete RPMI): 1640 RPMI plus bicarbonate, lOOu/ml
penicillin/streptomycin, 2mM glutamine, 2x10-^ M 2- mercaptoethanol, 1 0% (w/v) heat inactivated foetal calf serum
DMEM (Dulbecco's Modified Eagles Medium): DMEM, lOOu/ml penicilin/streptomycin, 2mM glutamine, 2x10-^ M 2-mercaptoethanol, 10% (w/v) heat inactivated foetal calf serum, 4.4% w/v) sodium bicarbonate, lOmM HEPES
HLl medium: H Ll, 30u/ml penicillin/streptomycin, 2mM glutamine, 2x10-^ M 2-mercaptoethanol
Table 2.0: Antibodies directed against mouse and hum an M HC class II molecules
Antibody Specificity Origin
L2 HLA-DQa chain Fermand et al.,1986
Genox 3.53 HLA-DQwl a and p chain Brodsky et ah,1979
M 5/114.15.2 H-2A (bdq) Bhattacharya e t a l , 1981
Tal4.1 HLA-DQwl P 10th HLA workshop
R1 HLA-DQwl, 4, 8 and 9 Johnson et a l, 1984 H U -11 HLA-DQwl, 3 and 4 Kasahara et a l, 1983 IIB3 HLA-DQwl, 4, 8, and 9 Koning et a l, 1985 HU -18 HLA-DQw3-like Ikeda et a l, 1984
HU-33 HLA-DQw5 Kasahara et a l , 1983
Leu 10 HLA-DQwl and DQw3 p Brodsky, 1984
Table 2.1: Antibodies directed against mouse Vp, CD4 and CDS molecules.
Antibody Specificity Origin
KT6-FITC CD4 Tomonari & Lovering 1988
KT-15-FITC CDS Tomonari & Spencer 1990
B20.6.5/BIO VP2 Finkel et al., 1989
KJ25 VP3 Pharmingen* KT4-10 VP4 Tomonari et a l, 1990 MR9-4 VP5.1,5.2 Pharmingen PR4-7 VP6 Pharmingen TR310 vp? Pharmingen F23.1 VPS.123 Staerz et a l, 1985 MR 10-2 Vp9 Pharmingen
KTIO v p io Tomonari & Lovering 1988
K i l l v p i i Tomonari & Lovering 1988
M Rll-1 Vpl2 Pharmingen
MR12.4 VP13 Zaller et a l, 1990
[*A11 Pharmingen antibodies are directly conjugated to PE.
2.2 METHODS
Mice and in vivo techniques
Generation o f transgenics
HLA transgenic mice were generated by co-injection of HLA a and P chain sub-cloned cDNAs into fertilised mouse oocytes by Dr. Colin Hetherington, following the method of Hogan et a l, (1986).
Mice
Young adult (6-8 weeks old) mice of strains FVB/N and C57BL/10 were obtained from the Specific Pathogen Free (SPF) unit of the Clinical Research Centre (CRC), Harrow. All mice were fed normal rodent laboratory diet and had access to drinking water ad libitum.
Immunisation o f mice
Peptides to be injected were solubilised in phosphate buffered saline (PBS) and mixed v/v with Complete Freund's Adjuvant (CFA). The resulting preparations were homogenised by sonication. 50p.l of the homogenate was injected into each of the hind foot pads of mice previously anaesthetised with lOOpl peritoneally injected CRC 'cocktail' (2ml Hypnovel [Roche] plus 4ml distilled water plus 2ml Hypnorm [Janssen] - in that order). Ovalbumin was injected at a final concentration of 1 mg/ml, the MBP and GAD peptides at 2mg/ml. Mice were than placed onto a heat pad during recovery from the anaesthetic.
Cellular immunological in vitro techniques
Cell lines
PGF (HLA-DQ6+) and WT51 (HLA-DQ3+) human B lymphoblastoid cell lines were manitained in 25cm^ or 75cm^ tissue culture flasks in complete RPMI and passaged every 3-4 days.
Untransfected L cells, HLA-DQ6+ L cells and HLA-DQ3+ L cells were maintained in complete DMEM. The HLA-DQ6+ cells were selected on 1 mg/ml Geneticin, and the -DQ3+ cells on 1 mg/ml HAT.
Generation o f T cell lines
Popliteal lymph node cells from immunised mice were removed at day 11 after immunisation and cultured in HLl medium at 2x10^ cells/ml in a 12 well plate (Costar Corp., Cambridge, MA). After a further 10 days they were restimulated using irradiated strain/HLA -m atched mouse splenocytes and peptide in RPMI (25|ig/m l final concentration). TCGF (1%) was added as an exogenous source of IL-2, 3 days after each round of stimulation. This cycle of restimulation was repeated a further three times. Lines were tested against a panel of APC 8-10 days after the previous restimulation.
Cell preparations
Spleen cells:
The appropriate mouse was sacrificed by cervical dislocation or carbon monoxide inhalation. The skin of the mouse was doused in 70% alcohol. The peritoneal cavity was opened and the spleen removed aseptically and placed in RPMI in a sterile Petri dish. Cells were dissociated from the splenic capsule using 19g needles attached to 1ml syringes or by placing the spleen in a sterile cell strainer and dissociating the cells with a sterile 5ml syringe plunger. The resulting suspension was filtered through a nylon mesh into a sterile
20ml universal tube, which was then centrifuged at 1,500rpm for 5 minutes. After washing and centrifugation the cell pellet was resuspended in complete RPMI.
Lymph node cells:
Popliteal lymph nodes were harvested aseptically and the cell suspensions prepared as for spleen. Mesenteric lymph nodes used in FACS analysis were removed aseptically and placed in 400|xl of FACS buffer in an eppendorf tube. The nodes were disrupted using a disposable pestle and the cell suspension filtered through a sterile nylon mesh.
In vitro proliferation assays
To measure activation/proliferation of lymphocytes to specific antigens, popliteal lymph node cells were removed from transgenic and non-transgenic, strain matched, control mice and prepared as outlined above. Cells were washed, centrifuged and resuspended at 1.6xlO^/ml in HLl medium. lOOjLil of cells was added to each well of a 96 well plate with PPD, peptide or medium alone (negative control). PPD was titrated at 100|ig/ml and 50|Lig/ml. Peptide was tested at 100|ig/ml, 50|Xg/ml and 25|ag/ml, cell numbers allowing. All tests were carried out in triplicate in a total volume of 200|xl/well. Cells were incubated at 37°C in a 5% CO2 incubator for 72 hours. After 3 days the wells were pulsed with 0.5uCi ^H-thymidine in l|il of complete RPMI 1640. The assay was harvested 6 hours later using the Betaplate system (Pharmacia) and 3H incorporation expressed as counts per minute. Data is presented as Acpm (ie. mean of triplicate samples minus medium alone background count).
In vitro restriction assays
Various cell lines were tested for antigen presentation to T cell lines, using 4x10^ APC plus 1x10^ T cells per well. The human B cell lines PGF and WT51; the MHC class II null mutant, RJ2.2.5; mock-transfected mouse L cells and their HLA-DQ transfected counterpart served as APC. All APC were treated with 40|ig/ml mitomycin c (Sigma) at
37^C for 40 min before washing and adding to microtitre plates. T cell line/APC assays were conducted in HLl medium for 72 hours as described above.
FACS analysis:
Antibody staining for v p analysis:
Mesenteric lymph nodes were removed as previously described and resuspended in FACS buffer (PBS, 1.0% BSA, 0.02% azide). Approximately 1x10^ cells were aliquoted/well of a 96 well plate and centrifuged at lOOOrpm for 1 minute. The cells were washed once more in FACS buffer and re-centrifuged. Each sample was then incubated with a saturating concentration (25pg/ml) of anti-CD4, -CDS and -Vp antibodies in a total volume of 50pl/well. FACS buffer alone acted as a negative control. After incubation at 4^C for 40 minutes the cells were given two further washes in FACS buffer. The cells were then incubated at 4^C for a further 20 minutes in a second layer of PE/S A (or FACS buffer for the directly conjugated Pharmingen antibodies). The cells were then washed twice before being analysed on a Becton Dickinson Facscan. Approximately 20,000 events were analysed for each sample.
All other staining procedures were performed as above using the appropriate antibodies and second layer reagents. In all cases specificty of antibody staining was determined by the use of either second layer only staining or an isotype matched negative control antibody. Antibody sources and specificities are outlined in tables 2.0 and 2.1
Immunocytochemistry
Frozen sections of transgenic and non-transgenic mouse thymus were fixed with acetone. Staining and photographs provided by D. Douek, RPMS.
M olecular biological techniques
Preparation o f DNA fragments fo r micro-injection
Those colonies identified as containing the correctly aligned cDNAs were used in the large scale preparation of recombinant pDOl-5/HLA-DQ cDNA stocks using the QIAGEN maxi-prep kit. The appropriate Nrul/Xbal fragments were then purified for microinjection by passing through a Nacs column as outlined in Chapter 3, section 3.2, ‘Generation of HLA-DQ transgenic mice’.
Restriction digest and isolation o f DNA from LMP gels
20-30|Lig of plasmid DNA was digested with the appropriate restriction enzyme(s) under
standard conditions and separated by electrophoresis through a 1.5% LMP agarose gel. The gel was viewed under long wave UV and the fragment of interest excised using a scalpel blade. The DNA fragment was purified by melting the agarose at 65°C for 5 minutes and, following the addition of an equal volume of TRIS was extracted with phenol and choroform. DNA was recovered after an overnight incubation at -20°C with 0.1% volume of 3M NaAc and 2 volumes of ethanol. After microfugation at 4°C for 15 minutes the DNA pellet was air dried and dissolved in -20|il of sterile distilled water. 0.1 volume was electrophoresed on a 0.7% agarose gel to verify the isolation of the fragment and to approximate the concentration against a known standard.
Ligation o f cDNA construct into pD Ol-5 vector
Approximately 20|Lig of pDOl-5 vector was digested with EcoRI restriction enzyme to generate compatible ends for ligation. The vector was dephosphorylated as described by Sambrook et ah, (1989) to prevent re-annealing. Briefly, after inactivation of restriction enzyme 2|xl of calf intestinal alkaline phosphatase (GIF) and xlO GIF buffer containing ImM ZnGl2, ImM MgGl2 and lOmM Tris-HGL pH8.3 was added to the linearised vector. For 5' overhangs the reaction was incubated for 30 mins at 37°G and, following the addition of a second aliquot of GIF, the incubation was repeated. The procedure for 3'
overhangs was similar, however, the second incubation was at 55^C for 45 mins.Vector was purified by electrophoresis through a 0.7% LMP agarose gel and the DNA was extracted as described previously.
Ligations generally contained 30ng of cut and dephosphorylated vector and 50-100ng of insert DNA in a 50)li1 reaction volume, containing Ix ligation buffer (50mM Tris-HCL pH7.6, lOmM M gCl], 5% w/v polyethylene glycol 8000 ImM ATP and ImM dithiotreitol) and 40 units of T4 DNA ligase at 15°C overnight.
Transformation o f E. Colt
5jil of the reaction mixture was added to lOOfxl of competent E. Coli D H 5aF in a 1.5ml eppendorf, incubated on ice for 1 hour and heat shocked at 42^C for 90 seconds. Following incubation at RT for 15 mins, 200|li1 of L broth was added and the cells grown at 37^C for
1 hour in a rotary shaker. 100p,l of the cell suspension was plated out onto LB-agar containing ampicillin and incubated 0 /N at 37^0. Antibiotic resistant colonies were transferred into 3ml of superbroth containing ampicillin and incubated O/N at 37®C in a rotary shaker. The cultures were stored at 4^C until needed for mini-preps.
Mini-preps
1.5ml of above culture was aliquoted into eppendorfs and centrifuged at 4®C for 30 seconds, resuspended in lOOp.1 of 50mM glucose, 25mM TRIS pHS, lOmM EDTA pHS. 200p,l of 1% SDS, 0.2M NaoH was added to each suspension and after mixing the lysates left on ice for 5 mins. 150p.l of 5M Ko Ac pH4.8 was then added to each sample and incubated on ice for a further 5 mins. After centrifuging at 1400rpm for 4 mins the supernatant was transferred to a fresh tube and the DNA precipitated by the addition of 450p.l isopropanol and cooling to -20C for at least 1 hour. The DNA was recovered by centrifugation at 1400rpm 10 mins, washed with 70% ethanol and dissolved in ~50p,l of water containing 20p.gml RNase. Restriction digests were used to confirm the integration and correct orientation of the insert.
Maxi-preps
A large scale preparation of the desired clones was carried out using the remainder of the 3ml culture, using Qiagen maxi-prep kits.
Isolation/purification o f DNA from tails and cell lines
Isolation of tail DNA
Individual tails were incubated at 55^C O/N in 700|il of tail buffer (50mM TRIS, lOOMm NaCl, lOOmM EDTA and 1% SDS) plus 35|il 50mg/ml Proteinase K. After vortexing,
700|Li1 phenol was added to each sample before spinning the suspension on a rotary mixer
for 40 mins. Aqueous phase and inter-phase removed after 10 min spin at 1200rpm and transferred to a new eppendorf. The suspension was rotated for a further 40 mins after the addition of 700)il of phenol/chloroform. The upper phase was again transferred after 10 min centrifugation and the DNA precipitated by addition 700|il isopropanol. Orange sticks' were used to pick up the DNA which was then rinsed in 70% ethanol and air-dried. Finally, DNA was dissolved in 500|li1 double distilled water.
Isolation of DNA from cell lines
Cell pellets (-6x10^ cells) were resuspended in 2mls of PBS, 2ml of lysis buffer and 12|Li1
Proteinase K. After mixing on a shaker for 1 hour the cell suspension was incubated at 37^C overnight. An equal volume of phenol was added to each sample and mixed by inversion, then on a rotating wheel for 30 minutes. After centrifugation at 2.5K for 5 minutes the top (aqueous) layer was decanted into a new 15ml tube. An equal volume of phenol was added to this aqueous phase and the process repeated. A final extraction with chloroform was followed by a standard Sodium acetate/ethanol precipitation step.
PCR methods
Using non-specific MHC class II primers:
250ng of DNA was amplified using 2.5pl of lOx PCR buffer (50mM KCL 0.1% TRITON-X-100, lOmM TRIS-HCL pH8.8, 60mM MgCl2), 125ng of each primer, 2mM
dNTPs, and 0.5U of Taq polymerase, plus ddw to a total volume of 25)il. The reaction mix was overlaid with mineral oil. Mg2+ concentration was titrated from 20-80mM final for each set of primers used. 30 cycles of PCR were carried out using a Techne PHC-2 Dri BlockR Cycler. A final incubation of 72^C for 8 mins was added to ensure that all extensions were complete. 5|Li1 of each PCR product was analysed on a 2% agarose gel. All pan HLA-DQ specific primers were used with 60mM MgCl, at 56°C for 1 min, 72°C for 30 seconds for 30 cycles. Allele specific primers were amplified for 30 cycles with 40mM MgCl at 55^C for 1 min, 72^C for 30 seconds. Sequences of the pan HLA-DQ and allele- specific primers are detailed in figs 3.3.1 and 3.3.2.
RT-PCR
For each tissue 5)ig RNA, in final volume of 21|l i1RNase-free water, was denatured by
heating to 95^C for 5 min, then cooled on ice for 5 min. 4|il lOxPCR buffer [500mM KCL, lOOmM TRIS pH8.4, 25mM MgCl% and 2mM dNTPs] 6|il RNase free water, 6|xl RNA guard, 5|xl AMV RT and 4|l i13' primer [500p.g/ml] was then added to the denatured
RNA. The mixture was incubated at RT for 10 mins and then at 42^C for 1 hour. At this point the reaction mix may be stored at -20^C. Using the appropriate set of nested primers a standard PCR was then carried out as previously described usiing 4\i\ of the RT mixture.
Southern blot analysis
20)Lig of genomic DNA was used for Southern blot analysis. DNA was digested O/N in a
50|li1 reaction volume using the appropriate restriction endonuclease under the conditons described by the manufacturer. Digestion of cosmid/plasmid controls was carried out for 2-5 hours. 7.H3 size standards and the digested products (containing 0.6 volume of sample buffer) were loaded onto a 0.7% agarose gel containing 0.5mg/ml EtBr and electrophoresed O/N at 25V in IxTAE buffer.
DNA was transferred to Hybond nylon membrane (Amersham) using the method of Southern. Briefly, agarose gels were denatured for 45 mins and neutralised 45 min (x2) before being blotted O/N in x20 SSC, as per Sambrook et a l, (1989). After transfer the